[Name of Document] APPLICATION FOR PATENT
[Identification Number] C1-A0320
[Filing Date] December 12, 2003
[Addressee] Esq. Commissioner of the Patent Office
[inventor]
[Address]
[ Name ]
[Inventor]
[Address]
[ Name ]
[Inventor]
[Address]
[ Name ]
[ Inventor]
[Address]
[Name]
[Applicant]
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA,
153-2, Nagai, Niihari-mura, Niihari-gun,
Ibaraki
TSUNODA, Hiroyuki
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA,
153-2, Nagai, Niihari-mura, Niihari-gun,
Ibaraki
NAKANO, Kiyotaka
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA,
153-2, Nagai, Niihari-mura, Niihari-gun,
Ibaraki
ORITA, Tetsuro
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA.
135, Komakado 1-chome, Gotenba-shi,
Shizuoka
TSUCHIYA, Masayuki
[Identification Number] 000003311
[Name or Appellation]
[Attorney]
CHUGAI SEIYAKU KABUSHIKI KAISHA
[Identification Number] 100102978
[Patent Attorney]
[Name or Appellation] Hatsushi Shimizu
[Nominated Attorney]
[Identification Number] 100108774
[Patent Attorney]
[Name or Appellation] Kazunori Hashimoto
[Payment]
[Registration Number]
[Amount]
[List of Attached Documents]
041092
21000 yen
[Name of Document] Claims
1
[Name of Document] Specification 1
[Name of Document] Drawings
[Name of Document] Abstract
1
1
[General Power of Attorney Number] 0216136
JP 2003-415746
[Document Name] Claims
[Claim 1]
An antibody comprising a single-chain polypeptide having binding activity against TPO
receptor (Mpl), wherein said antibody comprises two heavy chain variable regions and two light
5 chain variable regions.
[Claim 2]
The antibody of claim 1 , wherein the two heavy chain variable regions and the two light
chain variable regions are arranged in the order of heavy chain variable region, light chain
variable region, heavy chain variable region, and light chain variable region from the N terminus
10 of the single-chain polypeptide.
[Claim 3]
The antibody of claim 1 or 2, wherein the two heavy chain variable regions and the two
light chain variable regions are linked by linkers.
[Claim 4]
15 The antibody of claim 3, wherein the linkers comprise 15 amino acids.
[Claim 5]
A chimeric antibody that binds to Mpl.
[Claim 6]
The antibody of claim 5, which is a humanized antibody.
20 [Claim 7]
The antibody of claim 5 or 6, which is a minibody.
[Claim 8]
An antibody that binds to soluble Mpl.
[Claim 9]
25 An antibody that binds to human Mpl and monkey Mpl.
[Claim 10]
An antibody having agonistic activity against human Mpl and monkey Mpl.
[Claim 11]
An antibody whose binding activity to soluble Mpl is KD = 1 0" 6 M or lower.
30 [Claim 12]
An antibody whose binding activity to soluble Mpl is KD =10" M or lower.
[Claim 13]
An antibody whose binding activity to soluble Mpl is KD = 10 M or lower.
[Claim 14]
35 An antibody whose TPO agonistic activity is EC50 = 100 nM or lower.
[Claim 15]
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An antibody whose TPO agonistic activity is EC50 = 30 nM or lower.
[Claim 16]
An antibody whose TPO agonistic activity is EC50 =10 nM or lower.
[Claim 17]
5 An antibody which comprises a heavy chain variable region, wherein said heavy chain
variable regions comprises CDR1 , CDR2 and CDR3 consisting of an amino acid sequence of
any one of:
[I] SEQ ED NOs: 3,4, and 5
[2] SEQ ID NOs: 6, 7, and 8
10 [3] SEQ ID NOs: 9, 10, and 1 1
[4] SEQ ID NOs: 12, 13, and 14
[5] SEQ ID NOs: 15, 16, and 17
[6] SEQ ID NOs: 18, 19, and 20
[7] SEQ ID NOs: 21, 22, and 23
1 5 [8] SEQ ID NOs: 24, 25, and 26
[9] SEQ ID NOs: 27, 28, and 29
[10] SEQ ID NOs: 30, 31, and 32
[II] SEQ ID NOs: 33, 34, and 35
[12] SEQ ID NOs: 36, 37, and 38
20 [13] SEQ ID NOs: 39, 40, and 41
[14] SEQ ED NOs: 42, 43, and 44
[15] SEQ ID NOs: 45, 46, and 47
[16] SEQ ID NOs: 48, 49, and 50
[17] SEQ ID NOs: 51, 52, and 53
25 [18] SEQ ID NOs: 54, 55, and 56
[19] SEQ ID NOs: 57, 58, and 59
[Claim 1 8]
An antibody which comprises a light chain variable region, wherein said light chain
variable region comprises CDR1, CDR2 and CDR3 consisting of an amino acid sequence of any
30 one of:
[1] SEQ ID NOs: 60, 61, and 62
[2] SEQ ID NOs: 63, 64, and 65
[3] SEQ ID NOs: 66, 67, and 68
[4] SEQ ID NOs: 69, 70, and 71
35 [5] SEQ ID NOs: 72, 73, and 74
[6] SEQ ID NOs: 75, 76, and 77
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[7] SEQ ID NOs: 78, 79, and 80
[8] SEQ ID NOs: 81, 82, and 83
[9] SEQ ED NOs: 84, 85, and 86
[10] SEQ ID NOs: 87, 88, and 89
5 [11] SEQ ID NOs: 90, 91, and 92
[12] SEQ ID NOs: 93, 94, and 95
[13] SEQ ID NOs: 96, 97, and 98
[14] SEQ ID NOs: 99, 100, and 101
[15] SEQ ID NOs: 102, 103, and 104
10 [16] SEQ ID NOs: 105, 106, and 107
[17] SEQ ID NOs: 108, 109, and 110
[18] SEQ ID NOs: 111, 112, and 113
[19] SEQ ID NOs: 114, 115, and 116.
[Claim 19]
1 5 An antibody that comprises a heavy chain variable region and a light chain variable
region of any one of:
[1] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 3, 4, and 5, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
20 of SEQ ID NOs: 60, 61, and 62;
[2] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 6, 7, and 8, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
25 [3] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 9, 10, and 11, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 66, 67, and 68;
[4] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
30 the amino acid sequences consisting of SEQ ID NOs: 12, 13, and 14, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 69, 70, and 71;
[5] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 15, 16, and 17, and a light chain variable
35 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 72, 73, and 74;
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[6] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 18, 19, and 20, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 75, 76, and 77;
5 [7] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 21, 22, and 23, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 78, 79, and 80;
[8] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
10 the amino acid sequences consisting of SEQ ID NOs: 24, 25, and 26, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 81, 82, and 83;
[9] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 27, 28, and 29, and a light chain variable
1 5 region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 84, 85, and 86;
[10] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 30, 3 1 , and 32, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
20 of SEQ ID NOs: 87, 88, and 89;
[11] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 33, 34, and 35, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 90, 91, and 92;
25 [12] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 36, 37, and 38, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 93, 94, and 95;
[13] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
30 the amino acid sequences consisting of SEQ ID NOs: 39, 40, and 41, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 96, 97, and 98;
[14] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 42, 43, and 44, and a light chain variable
35 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 99, 100, and 101;
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[15] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 45, 46, and 47, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 102, 103, and 104;
5 [16] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 48, 49, and 50, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 105, 106, and 107;
[17] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
10 the amino acid sequences consisting of SEQ ID NOs: 5 1 , 52, and 53, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 108, 109, and 110;
[18] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 54, 55, and 56, and a light chain variable
1 5 region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 111, 112, and 113;
[19] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 57, 58, and 59, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
20 of SEQ ID NOs: 114, 115, and 116.
[Claim 20]
An antibody that comprises a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 1 1 8.
[Claim 21]
25 An antibody that comprises a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 120.
[Claim 22]
An antibody that comprises a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 118 and a light chain variable region comprising the amino acid
30 sequence of SEQ ID NO: 120.
[Claim 23]
An antibody comprising the amino acid sequence of SEQ ID NO: 122.
[Claim 24]
An antibody comprises the amino acid sequence of SEQ ID NO: 2.
35 [Claim 25]
An antibody having an activity equivalent to that of an antibody of any one of claims 1 7
JP 2003-415746
to 24, wherein said antibody comprises the amino acid sequence set forth in any one of claims 17
to 24, in which one or more amino acids have been substituted, deleted, added and/or inserted.
[Claim 26]
An antibody that recognizes an epitope recognized by an antibody of any one of claims
5 17 to 25.
[Claim 27]
An antibody that recognizes the region of amino acids 26 to 274 of human Mpl.
[Claim 28]
An antibody of any one of claims 1 to 27, which has TPO agonistic activity.
10 [Claim 29]
A polynucleotide encoding an antibody of any one of claims 1 to 28.
[Claim 30]
A polynucleotide hybridizing to the polynucleotide of claim 29 under stringent
conditions, wherein said polynucleotide encodes an antibody having activity equivalent to that of
15 an antibody of any one of claims 1 to 28.
[Claim 31]
A vector comprising the polynucleotide of claim 29 or 30.
[Claim 32]
A host cell that carries the polynucleotide of claim 29 or 30, or the vector of claim 3 1 .
20 [Claim 33]
A pharmaceutical composition comprising an antibody of any one of claims 1 to 28.
7
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[Document Name] Specification
[Title of the Invention] ANTI-MPL ANTIBODIES
[Technical Field]
5 [0001]
The present invention relates to anti-Mpl antibodies.
[Background of the Invention]
[0002]
Thrombopoietin (TPO) is a factor that enhances the differentiation and maturation of
10 megakaryocytes (platelet precursor cells) from hemopoietic stem cells into platelets. TPO also
functions as a cytokine with an important role in the regulation of platelet number. TPO is
converted into its active form through the cleavage of a TPO precursor comprising 353 amino
acids.
Mpl is a TPO receptor, and human Mpl molecules are known to exist in two forms
15 comprising 572 and 635 amino acids. The human Mpl gene sequence has already been
analyzed (see Non-patent Document 1 and GenBank accession No. NM_005373).
Most cytokine receptors dimerize upon ligand binding, and transduce signals into cells.
It has been reported that TPO similarly binds to its own specific receptor MPL, which leads to
dimerization of the receptor, thereby transducing signals into cells and exerting physiological
20 effects (see Non-patent Document 2).
Antibodies exhibiting agonistic activity have been reported among those antibodies that
bind to receptors having the above features.
For example, an antibody against the erythropoietin (EPO) receptor has been reported to
substitute for erythropoietin function. The monovalent form (Fab) of the antibody is capable of
25 binding to the EPO receptor but is unable to transduce signals. Thus, dimerization of the
erythropoietin receptor via bivalent binding is assumed to be essential for signal transduction
(see Non-patent Document 3).
Antibodies that bind to Mpl and exhibit TPO agonistic activity have also been reported
(see Non-patent Documents 4 and 5). This suggests that receptor dimerization is induced upon
30 binding of a bivalent antibody with regards to MPL as well.
Meanwhile, a single-chain antibody (scFv) has been reported to exhibit TPO agonistic
activity (see Patent Document 1). However, it has been revealed that, the underlying
mechanism of scFv exhibiting TPO agonistic activity is that a part of scFv dimerizes (diabody)
and this diabody becomes the actual active unit (see Patent Documents 2 to 4).
35 [0003]
[Patent Document 1] US Patent No. 6342220
8 JP 2003-415746
[Patent Document 2] WO 01/79494
[Patent Document 3] WO 02/33072
[Patent Document 4] WO 02/33073
[Non-patent Document 1] Palacios et aL, 1985, Cell, 41, 727 '-73 4
5 [Non-patent Document 2] Souyriefa/., 1990, Cell, Vol.63, 1137-1147
[Non-patent Document 3]Elliott, S. et aL, 1996, J. Biol. Chem., 271(40), 24691-24697
[Non-patent Document 4] Abe et aL, 1998, Immunol. Lett., 61, 73-78
[Non-patent Document 5] Bijia Deng et aL, 1998, Blood, 92, 1981-1988
[Disclosure of the Invention]
10 [Problems to be Solved by the Invention]
[0004]
The present invention was achieved in view of the above circumstances. An objective
of the present invention is to provide novel anti-Mpl antibodies having TPO agonistic activity.
[Means for Solving the Problems]
15 [0005]
The present inventors performed exhaustive research to solve the above objective. The
present inventors prepared and purified anti-human Mpl antibody VB22B, and established a
single-chain antibody expression system using genetic engineering techniques. Specifically, the
variable region of anti-human Mpl antibody was first cloned, and a diabody expression vector
20 pCXND3-VB22B db for the anti-human Mpl antibody was prepared. This pCXND3-VB22B
db vector was then used to generate an expression vector pCXND3-VB22B sc(Fv)2 for
anti-human Mpl antibody sc(Fv)2. Anti-human Mpl sc(Fv)2 was transiently expressed in
CHO-DG44 cells using the expression vector pCXND3-VB22B sc(Fv) 2 , and then purified from
the culture supernatant. In control experiments, VB22B diabody was transiently expressed in
25 COS7 cells using the above pCXND3-VB22B db vector, and then purified from the culture
supernatant.
[0006]
In addition, VB22B diabody and VB22B sc(Fv) 2 were evaluated for their TPO-like
agonistic activities. The results showed that VB22B diabody and VB22B sc(Fv) 2 exhibit higher
30 agonistic activities compared to VB22B IgG, and thus activities equivalent to or higher than that
of the natural ligand, human TPO.
[0007]
More specifically, the present invention relates to:
(1) an antibody comprising a single-chain polypeptide having binding activity against
35 TPO receptor (Mpl), wherein said antibody comprises two heavy chain variable regions and two
light chain variable regions;
JP 2003-415746
10
(2) the antibody of (1), wherein the two heavy chain variable regions and the two light
chain variable regions are arranged in the order of heavy chain variable region, light chain
variable region, heavy chain variable region, and light chain variable region from the N terminus
of the single-chain polypeptide;
(3) the antibody of (1) or (2), wherein the two heavy chain variable regions and the two
light chain variable regions are linked by linkers;
(4) the antibody of (3), wherein the linkers comprise 1 5 amino acids;
(5) a chimeric antibody that binds to Mpl;
(6) the antibody of (5), which is a humanized antibody;
(7) the antibody of (5) or (6), which is a minibody;
(8) an antibody that binds to soluble Mpl;
(9) an antibody that binds to human Mpl and monkey Mpl;
15
20
(
(
(
(
(
(
(
(
0
1
2
3
4
5
6
7
an antibody having agonistic activity against human Mpl and monkey Mpl;
an antibody whose binding activity to soluble Mpl is KD = 1 0" 6 M or lower;
an antibody whose binding activity to soluble Mpl is KD = 10 M or lower;
an antibody whose binding activity to soluble Mpl is KD =10" M or lower;
an antibody whose TPO agonistic activity is EC50 = 100 nM or lower;
an antibody whose TPO agonistic activity is EC50 = 30 nM or lower;
an antibody whose TPO agonistic activity is EC50 =10 nM or lower;
an antibody which comprises a heavy chain variable region, wherein said heavy
e regions comprises CDR1, CDR2 and CDR3 consisting of an amino acid sequence
25
30
35
chain variab
of any one of:
[I] SEQ ID NOs: 3, 4, and 5
[2] SEQ ID NOs: 6, 7, and 8
[3] SEQ ID NOs: 9, 10, and 11
[4] SEQ ID NOs: 12, 13, and 14
[5] SEQ ID NOs: 15, 16, and 17
[6] SEQ ID NOs: 18, 19, and 20
[7] SEQ ID NOs: 21, 22, and 23
[8] SEQ ID NOs: 24, 25, and 26
[9] SEQ ID NOs: 27, 28, and 29
[10] SEQ ID NOs: 30, 31, and 32
[II] SEQ ID NOs: 33, 34, and 35
[12] SEQ ID NOs: 36, 37, and 38
[13] SEQ ID NOs: 39, 40, and 41
[14] SEQ ID NOs: 42, 43, and 44
10 JP 2003-415746
[15] SEQ ID NOs: 45, 46, and 47
[16] SEQ ID NOs: 48, 49, and 50
[17] SEQ ID NOs: 51,52, and 53
[18] SEQ ID NOs: 54, 55, and 56
5 [19] SEQ ID NOs: 57, 58, and 59;
(18) an antibody which comprises a light chain variable region, wherein said light chain
variable region comprises CDR1, CDR2 and CDR3 consisting of an amino acid sequence of any
one of:
[I] SEQ ID NOs: 60, 61, and 62
10 [2] SEQ ID NOs: 63, 64, and 65
[3] SEQ ID NOs: 66, 67, and 68
[4] SEQ ID NOs: 69, 70, and 71
[5] SEQ ID NOs: 72, 73, and 74
[6] SEQ ID NOs: 75, 76, and 77
1 5 [7] SEQ ID NOs: 78, 79, and 80
[8] SEQ ID NOs: 81, 82, and 83
[9] SEQ ED NOs: 84, 85, and 86
[10] SEQ ID NOs: 87, 88, and 89
[II] SEQ ID NOs: 90, 91, and 92
20 [12] SEQ ID NOs: 93, 94, and 95
[13] SEQ ID NOs: 96, 97, and 98
[14] SEQ ID NOs: 99, 100, and 101
[15] SEQ ID NOs: 102, 103, and 104
[16] SEQ ID NOs: 105, 106, and 107
25 [17] SEQ ID NOs: 108, 109, and 110
[18] SEQ ID NOs: 111, 112, and 113
[19] SEQ ID NOs: 114, 115, and 116;
(19) an antibody that comprises a heavy chain variable region and a light chain variable
region of any one of:
30 [1] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 3, 4, and 5, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 60, 61, and 62;
[2] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
35 the amino acid sequences consisting of SEQ ID NOs: 6, 7, and 8, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
11 JP 2003-415746
of SEQ ID NOs: 63, 64, and 65;
[3] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 9, 10, and 11, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
5 of SEQ ID NOs: 66, 67, and 68;
[4] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 12, 13, and 14, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 69, 70, and 71;
10 [5] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 15, 16, and 17, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 72, 73, and 74;
[6] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
15 the amino acid sequences consisting of SEQ ID NOs: 18, 19, and 20, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 75, 76, and 77;
[7] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 21, 22, and 23, and a light chain variable
20 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 78, 79, and 80;
[8] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 24, 25, and 26, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
25 of SEQ ID NOs: 8 1 , 82, and 83 ;
[9] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 27, 28, and 29, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 84, 85, and 86;
30 [10] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 30, 3 1, and 32, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 87, 88, and 89;
[11] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
35 the amino acid sequences consisting of SEQ ID NOs: 33, 34, and 35, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
12 JP 2003-415746
of SEQ ID NOs: 90, 91, and 92;
[12] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 36, 37, and 38, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
5 of SEQ ID NOs: 93, 94, and 95;
[13] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 39, 40, and 41, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 96, 97, and 98;
10 [14] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 42, 43, and 44, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 99, 100, and 101;
[15] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
15 the amino acid sequences consisting of SEQ ID NOs: 45, 46, and 47, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 102, 103, and 104;
[16] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 48, 49, and 50, and a light chain variable
20 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 105, 106, and 107;
[17] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 51, 52, and 53, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
25 of SEQ ID NOs: 108, 109, and 110;
[18] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 54, 55, and 56, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
ofSEQIDNOs: 111, 112, and 113;
30 [19] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 57, 58, and 59, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 114, 115, and 116;
(20) an antibody that comprises a heavy chain variable region comprising the amino
35 acid sequence of SEQ ID NO: 1 1 8;
(21) an antibody that comprises a light chain variable region comprising the amino acid
13
JP 2003-415746
sequence of SEQ ID NO: 120;
(22) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 118 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 120;
(23) an antibody comprising the amino acid sequence of SEQ ID NO: 122;
(24) an antibody comprises the amino acid sequence of SEQ ID NO: 2;
(25) an antibody having an activity equivalent to that of an antibody of any one of (17)
to (24), wherein said antibody comprises the amino acid sequence set forth in any one of (17) to
(24), in which one or more amino acids have been substituted, deleted, added and/or inserted;
(26) an antibody that recognizes an epitope recognized by an antibody of any one of
(17) to (25);
(27) an antibody that recognizes the region of amino acids 26 to 274 of human Mpl;
(28) an antibody of any one of (1) to (27), which has TPO agonistic activity;
(29) a polynucleotide encoding an antibody of any one of (1) to (28);
(30) a polynucleotide hybridizing to the polynucleotide of (29) under stringent
conditions, wherein said polynucleotide encodes an antibody having activity equivalent to that of
an antibody of any one of (1) to (28);
(31) a vector comprising the polynucleotide of (29) or (30);
(32) a host cell that carries the polynucleotide of (29) or (30), or the vector of (31); and
(33) a pharmaceutical composition comprising an antibody of any one of (1) to (28).
[Effects of the Invention]
[0008]
Recombinant human TPOs have been tested as therapeutic agents for
chemotherapy- induced thrombocytopenia in various clinical trials. In these trials, a major
problem that was reported was the production of anti-TPO antibody in TPO-treated patients
(Junzhi Li, et al., Blood (2001) 98: 3241-324; Saroj Vandhan-Raj, et al, Ann. Intern. Med.
(2000) 132: 364-368). Specifically, production of neutralizing antibodies that inhibit
endogenous TPO activity, and the resulting onset of thrombocytopenia, were reported.
Administration of agonistic minibodies (low molecular weight antibodies) against anti-TPO
receptor of the present invention does not induce the production of antibodies against
endogenous TPO. Furthermore, since antibody miniaturization results in increased specific
activity and short half-life in blood, the effective concentration in blood can be easily regulated,
presenting a further clinical advantage. Accordingly, the present antibodies are expected to be
therapeutic agents for thrombocytopenia which are more effective than naturally-occurring TPO
or its agonistic antibodies. Furthermore, since minibodies are not glycosylated, expression
systems for expressing the recombinant proteins are not limited, and minibodies can be produced
JP 2003-415746
in any expression system such as cell lines from mammals, yeast, insect cells, and E. coli.
Since binding specificity against mutant TPO receptor is different from that of TPO, minibodies
are expected to bind specific mutants and show agonistic activity against mutated TPO receptor
detected in the CAMT patients, who genetically have mutated TPO receptor and develop
5 thrombocytopenia.
[Best Mode for Carrying Out the Invention]
[0009]
The present invention provides antibodies that bind to the TPO receptor (Mpl).
The antibodies of the present invention comprise all types of antibodies, including
10 antibodies with modified amino acid sequences, such as minibodies, humanized antibodies, and
chimeric antibodies; antibodies that have been modified by binding with other molecules (for
example, polymers such as polyethylene glycol); and antibodies whose sugar chains have been
modified.
It is preferable that the antibodies of the present invention have agonistic activity against
15 Mpl.
[0010]
In a preferred embodiment, the antibodies of the present invention comprise, for
example, minibodies.
The minibodies comprise antibody fragments lacking portions of the whole antibody
20 (for example, whole IgG). The minibodies are not particularly limited as long as they have
binding activity to their antigens. The minibodies of the present invention have markedly
higher activities compared to their corresponding whole antibodies. There are no particular
limitations on the antibody fragments of the present invention as long as they are portions of the
whole antibody, and preferably contain heavy chain variable regions (VH) and/or light chain
25 variable regions (VL). The amino acid sequences of VH or VL may contain substitutions,
deletions, additions and/or insertions. Furthermore, the antibody fragment may also lack
portions of VH or/and VL, as long as it has binding ability to its antigen. In addition, the
variable regions may be chimerized or humanized. Such antibody fragments include, for
example, Fab, Fab', F(ab')2, and Fv. An example of a minibody includes Fab, Fab', F(ab')2, Fv,
30 scFv (single-chain Fv), diabody, and sc(Fv)2 (single-chain (Fv)2).
[0011]
Herein, an "Fv" fragment is the smallest antibody fragment and contains a complete
antigen recognition site and a binding site. The "Fv" fragment is a dimer (VH-VL dimer) in
which a single VH and a single VL are strongly linked by a non-covalent bond. The three
35 complementarity-determining regions (CDRs) of each of the variable regions interact with each
other to form an antigen-binding site on the surface of the VH-VL dimer. Six CDRs confer the
15
JP 2003-415746
antigen-binding site of an antibody. However, a single variable region (or a half of Fv
containing only three CDRs specific to an antigen) alone is also capable of recognizing and
binding an antigen although its affinity is lower than the affinity of the entire binding site.
[0012]
5 scFv contains the VH and VL regions of an antibody, and these regions exist on a single
polypeptide chain. Generally, an Fv polypeptide further contains a polypeptide linker between
VH and VL, and therefore an scFv can form a structure required for antigen binding. See,
Pluckthun "The Pharmacology of Monoclonal Antibodies" Vol. 113 (Rosenburg and Moore eds.
(Springer Verlag, New York, pp.269-3 1 5, 1994) for the review of scFv. In the present invention,
1 0 linkers are not especially limited as long as they do not inhibit expression of antibody variable
regions linked at both ends of the linkers.
[0013]
The term "diabody" refers to a bivalent antibody fragment constructed by gene fusion
(Holliger P et aL, Proc. Natl. Acad. Sci. USA (1993) 90: 6444-6448; EP 404,097; WO 93/11161
15 and others). Diabodies are dimers comprising two polypeptide chains, where each polypeptide
chain comprises a VL and a VH connected with a linker short enough to prevent interaction of
these two domains, for example, a linker of about five residues. The VL and VH encoded on
the same polypeptide chain will form a dimer because the linker between them is too short to
form a single-chain variable region fragment. As a result, the polypeptide chains form a dimer,
20 and thus the diabody has two antigen binding sites.
[0014]
sc(Fv) 2 is a single-chain minibody produced by linking two units of VH and two units of
VL with linkers and such (Hudson et al. 9 J Immunol. Methods (1999) 231: 177-189). sc(Fv) 2
exhibits a particularly high agonistic activity compared to the whole antibody and other
25 minibodies. sc(Fv) 2 can be produced, for example, by linking two scFv molecules.
[0015]
In a preferable antibody, the two VH units and two VL units are arranged in the order of
VH, VL, VH, and VL (|VH]-linker-|yL]-linker-[VH]-linker-|yL]) beginning from the N
terminus of a single-chain polypeptide.
30 The order of the two VH units and two VL units is not limited to the above arrangement,
and they may be arranged in any order. Examples of the arrangements are listed below.
|VL]-linker-[VH]-linker-|yH]-linker-[VL]
[VH]-linker-[VL]-linker-|yL]-linker-[VH]
|VH]-linker-|VHHin^
35 |VL]-linker-(yL]-lin^
|VL]-linker-|yH]-linker-|yL]-linker-[VH]
16 JP 2003-415746
[0016]
The linkers to be used for linking the variable regions of an antibody comprise arbitrary
peptide linkers that can be introduced by genetic engineering, synthetic linkers, and linkers
disclosed in, for example, Holliger, P. et at, Protein Engineering (1996) 9 (3): 299-305.
5 Peptide linkers are preferred in the present invention. There are no limitations as to the length
of the peptide linkers. The length can be selected accordingly by those skilled in the art
depending on the purpose, and is typically 1-100 amino acids, preferably 3-50 amino acids, more
preferably 5-30 amino acids, and even more preferably 12-18 amino acids (for example, 15
amino acids).
10 [0017]
For example, such peptide linkers include:
Ser
Gly Ser
Gly Gly Ser
15 Ser Gly Gly
Gly Gly Gly Ser
Ser Gly Gly Gly
Gly Gly Gly Gly Ser
Ser Gly Gly Gly Gly
20 Gly Gly Gly Gly Gly Ser
Ser Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Ser
Ser Gly Gly Gly Gly Gly Gly
(Gly Gly Gly Gly Ser) n
25 (Ser Gly Gly Gly Q\y\
where n is an integer of 1 or larger. The lengths and sequences of peptide linkers can
be selected accordingly by those skilled in the art depending on the purpose.
[0018]
In an embodiment of the present invention, a particularly preferable sc(Fv)2 includes,
30 for example, the sc(Fv)2 below.
[VH] -peptide linker (15 amino acids)- [VL] -peptide linker (15 amino
acids)-[VH]-peptide linker (15 amino acids)-[VL]
[0019]
Synthetic linkers (chemical crosslinking agents) include crosslinking agents routinely
35 used to crosslink peptides, for example, N-hydroxy succinimide (NHS), disuccinirnidyl suberate
(DSS), bis(sulfosuccinimidyl) suberate (BS ), dithiobis(succinimidyl propionate) (DSP),
JP 2003-415746
dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate)
(EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate
(DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]
sulfone (BSOCOES), and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone
5 (sulfo-BSOCOES). These crosslinking agents are commercially available.
[0020]
In general, three linkers are required to link four antibody variable regions together.
The linkers to be used may be of the same type or different types. In the present invention, a
preferable minibody is a diabody, even more preferably, an sc(Fv)2. Such a minibody can be
10 prepared by treating an antibody with an enzyme, for example, papain or pepsin, to generate
antibody fragments, or by constructing DNAs encoding those antibody fragments and
introducing them into expression vectors, followed by expression in an appropriate host cell (see,
for example, Co, M. S. et aL, J. Immunol. (1994) 152: 2968-2976; Better, M. and Horwitz, A. H.,
Methods Enzymol. (1989) 178: 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol.
15 (1989) 178: 497-515; Lamoyi, E., Methods Enzymol. (1986) 121: 652-663; Rousseaux, J. etal,
Methods Enzymol. (1986) 121: 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol.
(1991)9: 132-137).
An antibody having exceedingly high agonistic activity can be prepared by reducing the
molecular weight of a full-length antibody, particularly by converting it into an sc(Fv)2.
20 [0021]
In a preferred embodiment, the antibodies of the present invention comprise modified
antibodies, such as chimeric antibodies and humanized antibodies that bind to Mpl. These
modified antibodies can be produced by known methods.
Chimeric antibodies are antibodies prepared by combining sequences derived from
25 different animal species, and include for example, antibodies comprising the heavy chain and
light chain variable regions of a murine antibody, and the heavy chain and light chain constant
regions of a human antibody. Chimeric antibodies can be prepared by known methods. For
example, a DNA encoding the V region of an antibody is linked to a DNA encoding the C region
of a human antibody, and the construct is inserted into an expression vector and introduced into a
30 host to produce chimeric antibodies.
Humanized antibodies are also referred to as "reshaped human antibodies". Such a
humanized antibody is obtained by transferring the complementarity-determining region (CDR)
of an antibody derived from a non-human mammal, for example mouse, to the
complementarity-determining region of a human antibody, and the general gene recombination
35 procedure for this is also known (see European Patent Application No. 125023 and WO
96/02576).
18
JP 2003-415746
Specifically, a DNA sequence designed to link a murine antibody CDR to the
framework region (FR) of a human antibody can be synthesized by PCR, using primers prepared
from several oligonucleotides containing overlapping portions of both CDR and FR terminal
regions (see methods described in WO 98/13388).
5 The human antibody framework region to be linked by CDR is selected in order to form
a favorable antigen-binding site in the complementarity-determining region. Amino acids of
the framework region in the antibody variable region may be substituted, as necessary, for the
complementarity-determining region of the reshaped human antibody to form a suitable
antigen-binding site (Sato, K. et al 9 Cancer Res. (1993) 53: 851-856).
10 The constant region of a human antibody is used as the C region of a chimeric antibody
or humanized antibody. For example, Cyl, Cy2, Cy3, and Cy4 can be used as the H chain, and
Ck and CA, can be used as the L chain. The human antibody C region may be modified to
improve the antibody or the stability of the antibody production.
Generally, chimeric antibodies comprise the variable region of an antibody from a
1 5 non-human mammal and the constant region derived from a human antibody. On the other
hand, humanized antibodies comprise the complementarity-determining region of an antibody
from a non-human mammal, and the framework region and C region derived from a human
antibody.
In addition, after a chimeric antibody or a humanized antibody is prepared, amino acids
20 in the variable region (for example, FR) and the constant region may be replaced with other
amino acids, and such.
The origin of the variable regions in chimeric antibodies or that of the CDRs in
humanized antibodies is not particularly limited, and may be derived from any type of animal.
For example, sequences of murine antibodies, rat antibodies, rabbit antibodies, camel antibodies
25 may be used.
In general, it is difficult to chimerize or humanize an antibody without losing the
agonistic activity of the original antibody. Nevertheless, the present invention succeeded in
preparing humanized antibodies having agonistic activity equivalent to that of the original
murine antibody.
30 Chimeric antibodies and humanized antibodies exhibit lower antigenicity in the human
body, and thus are expected to be useful when administered to humans for therapeutic purposes.
[0022]
In one embodiment, the preferred antibodies of the present invention include antibodies
that bind to soluble Mpl. The term "soluble Mpl" herein refers to Mpl molecules excluding
35 those expressed on the cell membrane. A specific example of a soluble Mpl is an Mpl lacking
the entire or a portion of the transmembrane domain. The transmembrane domain of human
19
JP 2003-415746
Mpl corresponds to amino acids 492 to 513 in SEQ ID NO: 123.
An antibody that binds to soluble recombinant Mpl can be used in detailed epitope
analysis and kinetic analysis of receptor-ligand binding, as well as for assessing the blood
concentration and dynamic behavior of the antibody in in vivo tests.
5 In one embodiment, the preferred antibodies of the present invention include antibodies
having binding activity against both human and monkey Mpl. Antibodies having agonistic
activity to both human and monkey Mpl are expected to be highly useful since the dynamic
behavior and in vivo effects of the antibody, which are generally difficult to determine in human
body, can be examined with monkeys.
1 0 The present invention also provides antibodies having agonistic activity to human Mpl
and monkey Mpl. Such antibodies may also have binding activity or agonistic activity against
Mpl from animals other than humans and monkeys (for example, mice).
In addition, the antibodies of the present invention include antibodies with TPO
agonistic activity (agonistic activity against Mpl) of EC50 = 100 nM or lower, preferably EC50 =
15 30 nM or lower, more preferably EC50 =10 nM or lower.
The agonistic activity can be determined by methods known to those skilled in the art,
for example, by the method described below.
The sequences for human Mpl (Palacios et al., Cell (1985) 41 : 727-734; GenBank Accession NO.
NM_005373), cynomolgus monkey Mpl (SEQ ID NO: 157), and mouse Mpl (GenBank
20 Accession NO. NM_0 10823) are already known.
In addition, the present invention includes antibodies whose binding activities to soluble
Mpl are KD = 10' 6 M or lower, preferably KD = 10' 7 M or lower, and more preferably KD = 10" 8
M or lower.
[0023]
25 In the present invention, whether the binding activity of an antibody to soluble
recombinant Mpl is KD = 10 M or lower can be determined by methods known to those skilled
in the art. For example, the activity can be determined using surface plasmon resonance with
Biacore. Specifically, soluble MPL-Fc protein is immobilized onto sensor chips. Reaction
rate constant can be determined by assessing the interaction between the antibody and the
30 soluble Mpl-Fc protein. The binding activity can be evaluated by ELISA (enzyme-linked
immunosorbent assays), EIA (enzyme immunoassays), RIA (radio immunoassays), or
fluorescent antibody techniques. For example, in enzyme immunoassays, a sample containing a
test antibody, such as purified antibody or culture supernatant of cells producing the test antibody,
is added to a plate coated with an antigen to which the test antibody can bind. After incubating
35 the plate with a secondary antibody labeled with an enzyme such as alkaline phosphatase, the
plate is washed and an enzyme substrate such as p-nitrophenyl phosphate is added. The
20
JP 2003-415746
antigen-binding activity can then be evaluated by determining the absorbance.
There is no specific limitation as to the upper limit of the binding activity; for example,
the upper limit may be set within a technically feasible range by those skilled in the art.
However, the technically feasible range may expand with the advancement of technology.
5 [0024]
In an embodiment, the preferred antibodies of the present invention include antibodies
recognizing epitopes that are recognized by any one of the antibodies indicated in (I) to (IX)
below. The antibody of any one of (I) to (IX) is preferably a minibody.
[0025]
10 (I)
Antibody comprising a VH that has CDR1 , 2, and 3 comprising the amino acid
sequences according to SEQ ID NOs in any one of (1) to (19) indicated below (name of each
antibody and the H chain CDR contained in the antibody are indicated inside the parentheses):
(I) SEQ ID NOs: 3, 4, and 5 (VA7: H chain CDR1, 2, and 3),
15 (2) SEQ ID NOs: 6, 7, and 8 (VA130: H chain CDR1, 2, and 3),
(3) SEQ ID NOs: 9, 10, and 11 (VA259: H chain CDR1, 2, and 3),
(4) SEQ ID NOs: 12, 13, and 14 (VB17B: H chain CDR1, 2, and 3),
(5) SEQ ID NOs: 15, 16, and 17 (VB12B: H chain CDR1, 2, and 3),
(6) SEQ ID NOs: 18, 19, and 20 (VB140: H chain CDR1, 2, and 3),
20 (7) SEQ ID NOs: 21, 22, and 23 (VB33: H chain CDR1, 2, and 3),
(8) SEQ ID NOs: 24, 25, and 26 (VB45B: H chain CDR1, 2, and 3),
(9) SEQ ID NOs: 27, 28, and 29 (V8B: H chain CDR1, 2, and 3),
(10) SEQ ID NOs: 30, 31, and 32 (VB115: H chain CDR1, 2, and 3),
(II) SEQ ID NOs: 33, 34, and 35 (V14B: H chain CDR1, 2, and 3),
25 (12) SEQ ID NOs: 36, 37, and 38 (V22B: H chain CDR1, 2, and 3),
(13) SEQ ID NOs: 39, 40, and 41 (VB16: H chain CDR1, 2, and 3),
(14) SEQ ID NOs: 42, 43, and 44 (VB157: H chain CDR1, 2, and 3),
(15) SEQ ID NOs: 45, 46, and 47 (VB4B: H chain CDR1, 2, and 3),
(16) SEQ ID NOs: 48, 49, and 50 (VB51: H chain CDR1, 2, and 3),
30 (17) SEQ ID NOs: 51, 52, and 53 (AB317: H chain CDR1, 2, and 3),
(18) SEQ ID NOs: 54, 55, and 56 (AB324: H chain CDR1, 2, and 3),
(19) SEQ ID NOs: 57, 58, and 59 (TA136: H chain CDR1, 2, and 3).
[0026]
(II)
35 Antibody comprising a VL which has CDR1 , 2, and 3 comprising the amino acid
sequences according to SEQ ID NOs in any one of (1) to (19) indicated below (name of each
21 JP 2003-415746
antibody and the L chain CDR in the antibody are indicated inside the parentheses):
(1) SEQ ID NOs: 60, 61, and 62 (VA7: L chain CDR1, 2, and 3),
(2) SEQ ID NOs: 63, 64, and 65 (VA130: L chain CDR1, 2, and 3),
(3) SEQ ID NOs: 66, 67, and 68 (VA259: L chain CDR1, 2, and 3),
5 (4) SEQ ID NOs: 69, 70, and 71 (VB17B: L chain CDR1, 2, and 3),
(5) SEQ ID NOs: 72, 73, and 74 (VB12B: L chain CDR1, 2, and 3),
(6) SEQ ID NOs: 75, 76, and 77 (VB140: L chain CDR1, 2, and 3),
(7) SEQ ID NOs: 78, 79, and 80 (VB33: L chain CDR1, 2, and 3),
(8) SEQ ID NOs: 81, 82, and 83 (VB45B: L chain CDR1, 2, and 3),
10 (9) SEQ ID NOs: 84, 85, and 86 (V8B: L chain CDR1 , 2, and 3),
(10) SEQ ID NOs: 87, 88, and 89 (VB 1 1 5 : L chain CDR1, 2, and 3),
(11) SEQ ID NOs: 90, 91, and 92 (V14B: L chain CDR1, 2, and 3),
(12) SEQ ID NOs: 93, 94, and 95 (VB22B: L chain CDR1, 2, and 3),
(13) SEQ ID NOs: 96, 97, and 98 (VB16: L chain CDR1, 2, and 3),
15 (14) SEQ ID NOs: 99, 100, and 101 (VB157: L chain CDR1, 2, and 3),
(15) SEQ ID NOs: 102, 103, and 104 (VB4B: L chain CDR1, 2, and 3),
(16) SEQ ID NOs: 105, 106, and 107 (VB51: L chain CDR1, 2, and 3),
(17) SEQ ID NOs: 108, 109, and 110 (AB317: L chain CDR1, 2, and 3),
(18) SEQ ID NOs: 111, 112, and 113 (AB324: L chain CDR1, 2, and 3),
20 (19) SEQ ID NOs: 114, 115, and 116 (TA136: L chain CDR1, 2, and 3).
[0027]
(III)
Antibody comprising a VH that comprises an amino acid sequence of the SEQ ID NO in
any one of (1) to (19):
25 (1) SEQ ID NO: 124 (VA7: VH),
(2) SEQ ID NO: 126 (VA130: VH),
(3) SEQ ID NO: 128 (VA259: VH),
(4) SEQ ID NO: 130 (VB17B: VH),
(5) SEQ ID NO: 132 (VB12B: VH),
30 (6) SEQ ID NO: 134 (VB140: VH),
(7) SEQ ID NO: 136 (VB33: VH),
(8) SEQ ID NO: 138 (VB45B: VH),
(9) SEQ ID NO: 140 (V8B: VH),
(10) SEQ ID NO: 142 (VB115: VH),
35 (11) SEQ ID NO: 144 (VB14B: VH),
(12) SEQ ID NO: 118 (VB22B: VH),
JP 2003-415746
(13) SEQ ID NO: 146 (VB16: VH),
(14) SEQ ID NO: 148 (VB157: VH),
(15) SEQ ID NO: 150 (VB4B: VH),
(16) SEQ ID NO: 152 (VB51: VH),
5 (17) SEQ ID NO: 155 (AB317: VH),
(18) SEQ ID NO: 159 (AB324: VH),
(19) SEQ ID NO: 162 (TA136: VH).
[0028]
(IV)
1 0 Antibody comprising a VL that comprises an amino acid sequence of the SEQ ID NO in
any one of (1) to (19):
(1) SEQ ID NO: 125 (VA7: VL),
(2) SEQ ID NO: 127 (VA130: VL),
(3) SEQ ID NO: 129 (VA259: VL),
15 (4) SEQ ID NO: 131 (VB17B: VL),
(5) SEQ ID NO: 133 (VB12B: VL),
(6) SEQ ID NO: 135 (VB140: VL),
(7) SEQ ID NO: 137 (VB33: VL),
(8) SEQ ID NO: 139 (VB45B: VL),
20 (9) SEQ ID NO: 141 (VB8B: VL),
(10) SEQ ID NO: 143 (VB115: VL),
(11) SEQ ID NO: 145 (VB14B: VL),
(12) SEQ ID NO: 120 (VB22B: VL),
(13) SEQ ID NO: 147 (VB16: VL),
25 (14) SEQ ID NO: 149 (VB157: VL),
(15) SEQ ID NO: 151 (VB4B: VL),
(16) SEQ ID NO: 153 (VB51: VL),
(17) SEQ ID NO: 157 (AB317: VL),
(18) SEQ ID NO: 161 (AB324: VL),
30 (19) SEQ ID NO: 163 (TA136: VL).
[0029]
(V)
Antibody comprising a VH and VL according to any one of (1 ) to (19):
(1) SEQ ID NOs: 3, 4, and 5 (VA7: H chain CDR1, 2, and 3); SEQ ID NOs: 60, 61, and 62
35 (VA7: LchainCDRl,2, and 3),
(2) SEQ ID NOs: 6, 7, and 8 (VA130: H chain CDR1, 2, and 3), SEQ ID NOs: 63, 64, and 65
23
JP 2003-415746
(VA130: L chain CDR1, 2, and 3),
(3) SEQ ID NOs: 9, 10, and 11 (VA259: H chain CDR1, 2, and 3); SEQ ID NOs: 66, 67, and 68
(VA259: L chain CDR1, 2, and 3),
(4) SEQ ID NOs: 12, 13, and 14 (VB17B: H chain CDR1, 2, and 3); SEQ ID NOs: 69, 70, and
5 71 (VB17B:LchainCDRl,2, and 3),
(5) SEQ ID NOs: 15, 16, and 17 (VB12B: H chain CDR1, 2, and 3); SEQ ID NOs: 72, 73, and
74 (VB12B: L chain CDR1, 2, and 3),
(6) SEQ ID NOs: 18, 19, and 20 (VB140: H chain CDR1, 2, and 3); SEQ ID NOs: 75, 76, and 77
(VB140: L chain CDR1, 2, and 3),
10 (7) SEQ ID NOs: 21, 22, and 23 (VB33: H chain CDR1, 2, and 3); SEQ ID NOs: 78, 79, and 80
(VB33: L chain CDR1, 2, and 3),
(8) SEQ ID NOs: 24, 25, and 26 (VB45B: H chain CDR1, 2, and 3); SEQ ID NOs: 81, 82, and
83 (VB45B: L chain CDR1, 2, and 3),
(9) SEQ ID NOs: 27, 28, and 29 (V8B: H chain CDR1, 2, and 3); SEQ ID NOs: 84, 85, and 86
1 5 (V8B: L chain CDR1 , 2, and 3),
(10) SEQ ID NOs: 30, 31, and 32 (VB115: H chain CDR1, 2, and 3); SEQ ID NOs: 87, 88, and
89 (VB115: L chain CDR1, 2, and 3),
(11) SEQ ID NOs: 33, 34, and 35 (V14B: H chain CDR1, 2, and 3); SEQ ID NOs: 90, 91, and 92
(VB14B: L chain CDR1, 2, and 3),
20 (12) SEQ ID NOs: 36, 37, and 38 (V22B: H chain CDR1, 2, and 3); SEQ ID NOs: 93, 94, and 95
(VB22B,: L chain CDR1 , 2, and 3),
(13) SEQ ID NOs: 39, 40, and 41 (VB16: H chain CDR1, 2, and 3); SEQ ID NOs: 96, 97, and 98
(VB16: L chain CDR1, 2, and 3),
(14) SEQ ID NOs: 42, 43, and 44 (VB157: H chain CDR1, 2, and 3); SEQ ID NOs: 99, 100, and
25 101 (VB157:LchainCDRl,2,and3),
(15) SEQ ID NOs: 45, 46, and 47 (VB4B: H chain CDR1, 2, and 3); SEQ ID NOs: 102, 103, and
104 (VB4B: L chain CDR1, 2, and 3),
(16) SEQ ID NOs: 48, 49, and 50 (VB51: H chain CDR1, 2, and 3); SEQ ID NOs: 105, 106, and
107 (VB51: L chain CDR1, 2, and 3),
30 (17) SEQ ID NOs: 51, 52, and 53 (AB317: H chain CDR1, 2, and 3); SEQ ID NOs: 108, 109,
and 1 1 0 (AB3 1 7: L chain CDR1 , 2, and 3),
(18) SEQ ID NOs: 54, 55, and 56 (AB324: H chain CDR1, 2, and 3); SEQ ID NOs: 111,112,
and 113 (AB324: L chain CDR1, 2, and 3),
(19) SEQ ID NOs: 57, 58, and 59 (TA136: H chain CDR1, 2, and 3); SEQ ID NOs: 114, 115, and
35 116(TA136: LchainCDRl, 2, and 3).
[0030]
JP 2003-415746
10
15
20
25
30
(VI)
Antibody comprising a VH and a VL that comprise the amino acid sequences according
to SEQ ID NOs in any one of (1) to (19) indicated below:
35
: 124 (VA7: VH), SEQ ID NO: 125 (VA7: VL),
: 126 (VA130: VH), SEQ ID NO: 127 (VA130: VL),
: 128 (VA259: VH), SEQ ID NO: 129 (VA259: VL),
: 130 (VB17B: VH), SEQ ID NO: 131 (VB17B: VL),
: 132 (VB12B: VH), SEQ ID NO: 133 (VB12B: VL),
: 134 (VB140: VH), SEQ ID NO: 135 (VB140: VL),
: 136 (VB33: VH), SEQ ID NO: 137 (VB33: VL),
: 138 (VB45B: VH), SEQ ID NO: 139 (VB45B: VL),
: 140 (V8B: VH), SEQ ID NO: 141 (VB8B: VL),
: 142(VB115: VH), SEQ ID NO: 143 (VB115: VL),
: 144 (VB14B: VH), SEQ ID NO: 145 (VB14B: VL),
: 118 (VB22B: VH), SEQ ID NO: 120 (VB22B: VL),
: 146 (VB16: VH), SEQ ID NO: 147 (VB16: VL),
: 148 (VB157: VH), SEQ ID NO: 149 (VB157: VL),
: 150 (VB4B: VH), SEQ ID NO: 151 (VB4B: VL),
: 152 (VB51: VH), SEQ ID NO: 153 (VB51: VL),
: 155 (AB317: VH), SEQ ID NO: 157 (AB317: VL),
18) SEQ ID NO: 159 (AB324: VH), SEQ ID NO: 161 (AB324: VL),
19) SEQ ID NO: 162 (TA136: VH), SEQ ID NO: 163 (TA136: VL).
[0031]
(VII)
Antibody comprising the amino acid sequence of SEQ ID NO: 122 (VB22B: scFv).
[0032]
(VIII)
Humanized antibody comprising an amino acid sequence according to any one of SEQ
ID NO: 2 (VB22B: sc(Fv) 2 ).
[0033]
(DQ
An antibody comprising an amino acid sequence of any one of (I) to (VIII) indicated
above, in which one or more amino acids have been substituted, deleted, added, and/or inserted,
wherein the antibody has activity equivalent to that of the antibody of any one of (I) to (V).
[0034]
Herein, the phrase "functionally equivalent" means that an antibody of interest has a
1) SEQ ID NO
2) SEQ ID NO
3) SEQ ID NO
4) SEQ ID NO
5) SEQ ID NO
6) SEQ ID NO:
7) SEQ ID NO
8) SEQ ID NO:
9) SEQ ID NO:
10) SEQ ID NO:
11) SEQ ID NO:
12) SEQ ID NO:
13) SEQ ID NO:
14) SEQ ID NO:
15) SEQ ID NO:
16) SEQ ID NO:
17) SEQ ID NO:
25
JP 2003-415746
biological or biochemical activity comparable to that of an antibody of the present invention.
Such activities include, for example, binding activities and agonistic activities.
[0035]
Methods for preparing polypeptides functionally equivalent to a certain polypeptide are
5 well known to those skilled in the art, and include methods of introducing mutations into
polypeptides. For example, those skilled in the art can prepare an antibody functionally
equivalent to the antibodies of the present invention by introducing appropriate mutations into
the antibody using site-directed mutagenesis (Hashimoto-Gotoh, T. et al Gene (1995) 152:
271-275; Zoller, MJ, and Smith, M. Methods Enzymol. (1983) 100: 468-500; Kramer, W. et al,
10 Nucleic Acids Res. (1984) 12: 9441-9456; Kramer, W. and Fritz HJ, Methods Enzymol. (1987)
154: 350-367; Kunkel, TA, Proc. Natl. Acad. Sci. USA (1985) 82: 488-492; Kunkel, Methods
Enzymol. (1988) 85: 2763-2766), or such. Amino acid mutations may occur naturally. Thus,
the present invention also comprises antibodies functionally equivalent to the antibodies of the
present invention and comprising the amino acid sequences of these antibodies, in which one or
15 more amino acids is mutated. Generally, the number of amino acids that are mutated is 50
amino acids or less, preferably 30 or less, more preferably 10 or less (for example, five amino
acids or less).
[0036]
An amino acid is preferably substituted for a different amino acid(s) that allows the
20 properties of the amino acid side-chain to be conserved. Examples of amino acid side chain
properties are: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V), hydrophilic amino acids
(R, D, N, C, E, Q, G, H, K, S, and T), amino acids comprising the following side chains: aliphatic
side chains (G, A, V, L, I, and P); hydroxyl-containing side chains (S, T, and Y);
sulfur-containing side chains (C and M); carboxylic acid- and amide-containing side chains (D,
25 N, E, and Q); basic side chains (R, K, and H); aromatic ring-containing side chains (H, F, Y, and
W) (amino acids are represented by one-letter codes in parentheses).
[0037]
A polypeptide comprising a modified amino acid sequence, in which one or more amino
acid residues is deleted, added, and/or replaced with other amino acids, is known to retain its
30 original biological activity (Mark, D. F. et al, Proc. Natl. Acad. Sci. USA (1984) 81: 5662-5666;
Zoller, M. J. & Smith, M. Nucleic Acids Research (1982) 10: 6487-6500; Wang, A. et al,
Science 224, 1431-1433; Dalbadie-McFarland, G et al, Proc. Natl. Acad. Sci. USA (1982) 79:
6409-6413).
[0038]
35 Fusion proteins containing antibodies that comprise the amino acid sequence of an
antibody of the present invention, in which two or more amino acid residues have been added,
26 JP 2003-415746
are included in the present invention. The fusion protein results from a fusion between one of
the above antibodies and a second peptide or protein, and is included in the present invention.
The fusion protein can be prepared by ligating a polynucleotide encoding an antibody of the
present invention and a polynucleotide encoding a second peptide or polypeptide in frame,
5 inserting this into an expression vector, and expressing the fusion construct in a host. Some
techniques known to those skilled in the art are available for this purpose. The partner peptide
or polypeptide to be fused with an antibody of the present invention may be a known peptide, for
example, FLAG (Hopp, T. P. etal, BioTechnology (1988) 6: 1204-1210), 6x His consisting of
six His (histidine) residues, lOx His, influenza hemagglutinin (HA), human c-myc fragment,
10 VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40 T antigen fragment, lck tag,
a-tubulin fragment, B-tag, Protein C fragment. Other partner polypeptides to be fused with the
antibodies of the present invention include, for example, GST (glutathione-S-transferase), HA
(influenza hemagglutinin), immunoglobulin constant region, P-galactosidase, and MBP
(maltose-binding protein). A polynucleotide encoding one of these commercially available
1 5 peptides or polypeptides can be fused with a polynucleotide encoding an antibody of the present
invention. The fusion polypeptide can be prepared by expressing the fusion construct.
[0039]
As described below, the antibodies of the present invention may differ in amino acid
sequence, molecular weight, isoelectric point, presence/absence of sugar chains, and
20 conformation depending on the cell or host producing the antibody, or purification method.
However, a resulting antibody is included in the present invention, as long as it is functionally
equivalent to an antibody of the present invention. For example, when an antibody of the
present invention is expressed in prokaryotic cells, for example E. coli, a methionine residue is
added to the N terminus of the original antibody amino acid sequence. Such antibodies are
25 included in the present invention.
[0040]
An antibody that recognizes an epitope recognized by the antibody according to any one
of (I) to (IX) indicated above is expected to have a high agonistic activity. Such antibodies can
be prepared by methods known to those skilled in the art. The antibody can be prepared by, for
30 example, determining the epitope recognized by the antibody according to any one of (I) to (DC)
by conventional methods, and using a polypeptide comprising one of the epitope amino acid
sequences as an immunogen. Alternatively, the antibody can be prepared by determining the
epitopes of conventionally prepared antibodies and selecting an antibody that recognizes the
epitope recognized by an antibody of any one of (I) to (IX).
35 [0041]
In the present invention, a particularly preferred antibody is an antibody that recognizes
JP 2003-415746
the epitope recognized by the antibody comprising the amino acid sequence of SEQ ID NO: 2.
The antibody comprising the amino acid sequence of SEQ ID NO: 2 is predicted to recognize the
region from Glu 26 to Leu 274, preferably the region from Ala 1 89 to Gly 245, more preferably
the region from Gin 213 to Ala 23 1 of human Mpl. Thus, antibodies recognizing the region of
5 amino acids 26 to 274, or amino acids 1 89 to 245, or amino acids 213 to 23 1 of human Mpl are
also included in the present invention.
[0042]
Antibodies recognizing regions of amino acids 26 to 274, amino acids 189 to 245, or
amino acids 213 to 231 of the human Mpl amino acid sequence (SEQ ID NO: 123) can be
1 0 obtained by methods known to those skilled in the art. Such antibodies can be prepared by, for
example, using a peptide comprising amino acids 26 to 274, amino acids 189 to 245, or amino
acids 213 to 231 of the human Mpl amino acid sequence (SEQ ID NO: 123) as an immunogen.
Alternatively, such antibodies can be prepared by determining the epitope of a conventionally
prepared antibody and selecting an antibody that recognizes the same epitope recognized by an
1 5 antibody of the present invention.
[0043]
The present invention provides antibodies described above in (I) to (IX). In an
embodiment of the present invention, a preferred antibody is the one shown in (V), a more
preferred antibody is the one shown in (VI), and a still more preferred is the one shown in (VII)
20 or (VIII).
[0044]
The present invention also provides vectors comprising polynucleotides encoding the
antibodies of the present invention, or polynucleotides which hybridize under stringent
conditions to the polynucleotides of the present invention and encode antibodies having activities
25 equivalent to those of the antibodies of the present invention. The polynucleotides of the
present invention are polymers comprising multiple bases or base pairs of deoxyribonucleic
acids (DNA) or ribonucleic acids (RNA), and are not particularly limited, as long as they encode
the antibodies of the present invention. They may also contain non-natural nucleotides. The
polynucleotides of the present invention can be used to express antibodies using genetic
30 engineering techniques. The polynucleotides of this invention can also be used as probes in the
screening of antibodies functionally equivalent to the antibodies of the present invention.
Specifically, DNAs that hybridize under stringent conditions to a polynucleotide encoding an
antibody of the present invention, and encode antibodies having activity equivalent to those of
the antibodies of the present invention can be obtained by techniques such as hybridization and
35 gene amplification (for example, PCR), using a polynucleotide of the present invention or a
portion thereof as a probe. Such DNAs are also included in the polynucleotides of the present
28
JP 2003-415746
invention. Hybridization techniques are well known to those skilled in the art (Sambrook, J et
ah, Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989). Such
hybridization conditions include, for example, conditions of low stringency. Examples of
conditions of low stringency include post-hybridization washing in 0.1 x SSC and 0.1% SDS at
5 42°C, and preferably in O.lx SSC and 0.1% SDS at 50°C. More preferable hybridization
conditions include those of high stringency. Highly stringent conditions include, for example,
washing in 5x SSC and 0.1% SDS at 65°C. In these conditions, the higher the temperature, the
higher the expectation of efficiently obtaining polynucleotides with a high homology. However,
several factors, such as temperature and salt concentration, can influence hybridization
1 0 stringency, and those skilled in the art can suitably select these factors to accomplish similar
stringencies.
[0045]
Antibodies that are encoded by polynucleotides obtained by the hybridization and gene
amplification techniques, and are functionally equivalent to the antibodies of the present
1 5 invention generally exhibit high homology to the antibodies of the this invention at the amino
acid level. The antibodies of the present invention include antibodies that are functionally
equivalent to the antibodies of the present invention, and exhibit high amino acid sequence
homology to the antibodies of this invention. The term "high homology" generally means
identity at the amino acid level of at least 50% or higher, preferably 75% or higher, more
20 preferably 85% or higher, still more preferably 95% or higher. Polypeptide homology can be
determined by the algorithm described in the report: Wilbur, W. J. and Lipman, D. J. Proc. Natl.
Acad. Sci. USA (1983) 80: 726-730.
[0046]
When E. coli is used as a host, there is no particular limitation as to the type of vector of
25 the present invention, as long as the vector contains an "ori" responsible for its replication in E.
coli and a marker gene. The "ori" ensures the amplification and mass production of the vector
in E. coli (for example, JM109, DH5a, HB101, and XLlBlue). The marker gene is used to
select the R coli transformants (for example, a drug resistance gene selected by an appropriate
drug such as ampicillin, tetracycline, kanamycin, and chloramphenicol). The vectors include,
30 for example, Ml 3 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. In addition to
the above vectors, for example, pGEM-T, pDIRECT, and pT7 can also be used for the
subcloning and excision of cDNAs.
[0047]
An expression vector is especially useful for the type of vectors of the present invention.
35 When an expression vector is expressed, for example, in E. coli, it should have the above
characteristics in order to be amplified in E. coli. Additionally, when E. coli, such as JM109,
29
JP 2003-415746
DH5a, HB101, or XLl-Blue are used as the host cell, the vector preferably has a promoter, for
example, lacZ promoter (Ward et al Nature (1989) 341: 544-546; FASEB J. (1992) 6:
2422-2427), araB promoter (Better et al, Science (1988) 240: 1041-1043), or T7 promoter, that
allows efficient expression of the desired gene in E. coli. Other examples of the vectors include
5 pGEX-5X-l (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (where BL21, a
strain expressing T7 RNA polymerase, is preferably used as the host).
[0048]
Furthermore, the vector may comprise a signal sequence for polypeptide secretion.
When producing polypeptides into the periplasm of E. coli, the pelB signal sequence (Lei, S. P.
10 et al. J. Bacteriol. (1987) 169: 4379) may be used as a signal sequence for polypeptide secretion.
For example, calcium chloride methods or electroporation methods may be used to introduce the
vector into a host cell.
[0049]
In addition to E. coli, expression vectors derived from mammals (e.g., pCDNA3
15 (Invitrogen), pEGF-BOS (Nucleic Acids Res. (1990) 18 (17): 5322), pEF, pCDM8), insect cells
(e.g., "Bac-to-BAC baculovirus expression system" (GIBCO-BRL), pBacPAK8), plants (e.g.,
pMHl, pMH2), animal viruses (e.g., pHSV, pMV, pAdexLcw), retroviruses (e.g., pZIPneo),
yeasts (e.g., "Pichia Expression Kit" (Invitrogen), pNVll, SP-Q01), and Bacillus subtilis (e.g.,
pPL608 5 pKTH50) may also be used as a vector of the present invention.
20 [0050]
In order to express proteins in animal cells such as CHO, COS, and NIH3T3 cells, the
vector preferably has a promoter necessary for expression in such cells, for example, an SV40
. promoter (Mulligan et al Nature (1979) 277: 108), MMLV-LTR promoter, EFla promoter
(Mizushima et al Nucleic Acids Res. (1990) 18: 5322), CMV promoter, etc.). It is even more
25 preferable that the vector also carries a marker gene for selecting transformants (for example, a
drug-resistance gene selected by a drug such as neomycin and G418). Examples of vectors
with such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13,
and such.
[0051]
30 In addition, to stably express a gene and amplify the gene copy number in cells, CHO
cells that are defective in the nucleic acid synthesis pathway are introduced with a vector
containing a DHFR gene (for example, pCHOI) to compensate for the defect, and the copy
number is amplified using methotrexate (MTX). Alternatively, a COS cell, which carries an
SV40 T antigen-expressing gene on its chromosome, can be transformed with a vector
35 containing the SV40 replication origin (for example, pcD) for transient gene expression. The
replication origin may be derived from polyoma virus, adenovirus, bovine papilloma virus
30
JP 2003-415746
(BPV), and such. Furthermore, to increase the gene copy number in host cells, the expression
vector may contain, as a selection marker, aminoglycoside transferase (APH) gene, thymidine
kinase (TK) gene, E. coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene,
dihydrofolate reductase (dhfr) gene, and such.
5 [0052]
Next, the vector is introduced into a host cell. The host cells into which the vector is
introduced are not particularly limited, for example, E. coli and various animal cells are available
for this purpose. The host cells may be used, for example, as a production system to produce
and express the antibodies of the present invention. In vitro and in vivo production systems are
10 available for polypeptide production systems. Production systems that use eukaryotic cells or
prokaryotic cells are examples of in vitro production systems.
[0053]
Eukaryotic cells that can be used are, for example, animal cells, plant cells, and fungal
cells. Known animal cells include: mammalian cells, for example, CHO (J. Exp. Med. (1995)
15 108: 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells such
asXenopus laevis oocytes (Valle, et al 9 Nature (1981) 291: 358-340), or insect cells (e.g., Sf9,
Sf2 1 , and Tn5). In the present invention, CHO-DG44, CHO-DXB 1 1 , COS7 cells, and BHK
cells can be suitably used. Among animal cells, CHO cells are particularly favorable for
large-scale expression. Vectors can be introduced into a host cell by, for example, calcium
20 phosphate methods, the DEAE-dextran methods, methods using cationic liposome DOTAP
(Boehringer-Mannheim), electroporation methods, lipofection methods.
[0054]
Plant cells include, for example, Nicotiana ta&acwm-derived cells known as a protein
production system. Calluses may be cultured from these cells. Known fungal cells include
25 yeast cells, for example, genus Saccharomyces such as Saccharomyces cerevisiae and
Saccharomyces pombe; and filamentous fungi, for example, genus Aspergillus such as
Aspergillus niger.
[0055]
Bacterial cells can be used in the prokaryotic production systems. Examples of
30 bacterial cells include E. coli (for example, JM109, DH5cx, HB101 and such); and Bacillus
subtilis.
[0056]
Next, the above host cells are cultured. Antibodies can be obtained by transforming
the cells with a polynucleotide of interest and in vitro culturing of these transformants.
35 Transformants can be cultured using known methods. For example, DMEM, MEM, RPMI
1640, or IMDM may be used as the culture medium for animal cells, and may be used with or
31 JP 2003-415746
without serum supplements such as FBS or fetal calf serum (FCS). Serum-free cultures are also
acceptable. The preferred pH is about 6 to 8 during the course of culturing. Incubation is
carried out typically at a temperature of about 30 to 40°C for about 15 to 200 hours. Medium is
exchanged, aerated, or agitated, as necessary.
5 [0057]
On the other hand, production systems using animal or plant hosts may be used as
systems for producing polypeptides in vivo. For example, a polynucleotide of interest is
introduced into an animal or plant and the polypeptide is produced in the body of the animal or
plant and then recovered. The "hosts" of the present invention includes such animals and
10 plants.
[0058]
Animals to be used for the production system include mammals or insects. Mammals
such as goats, pigs, sheep, mice, and cattle may be used (Vicki Glaser SPECTRUM
Biotechnology Applications (1993)). Alternatively, the mammals may be transgenic animals.
15 For example, a polynucleotide of interest is prepared as a fusion gene with a gene
encoding a polypeptide specifically produced in milk, such as the goat P -casein gene. DNA
fragments containing the fusion gene are injected into goat embryos, which are then introduced
back to female goats. The desired antibody can be obtained from milk produced by the
transgenic goats, which are bom from the goats that received the embryos, or from their
20 offspring. Appropriate hormones may be administered to increase the volume of milk
containing the antibody produced by the transgenic goats (Ebert, K.M. et ai, Bio/Technology
(1994) 12: 699-702).
[0059]
Insects, such as silkworms, may also be used. Baculoviruses carrying a polynucleotide
25 encoding an antibody of interest can be used to infect silkworms, and the antibody of interest can
be obtained from the body fluids (Susumu, M. et aL 9 Nature (1985) 315: 592-594).
[0060]
Plants used in the production system include, for example, tobacco. When tobacco is
used, a polynucleotide encoding an antibody of interest is inserted into a plant expression vector,
30 for example, pMON 530, and then the vector is introduced into a bacterium, such as
Agrobacterium tumefaciens. The bacteria are then used to infect tobacco such as Nicotiana
tabacum, and the desired antibodies can be recovered from the leaves (Julian K.-C. Ma et al. 9 Eur.
J. Immunol. (1994) 24: 131-138).
[0061]
35 The resulting antibody may be isolated from the inside or outside (such as the medium)
of host cells, and purified as a substantially pure and homogenous antibody. Methods are not
32 JP 2003-415746
limited to any specific method and any standard method for isolating and purifying antibodies
may be used. Polypeptides may be isolated and purified, by selecting an appropriate
combination of, for example, chromatographic columns, filtration, ultrafiltration, salting out,
solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide
5 gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and others.
Chromatographies include, for example, affinity chromatographies, ion exchange
chromatographies, hydrophobic chromatographies, gel filtrations, reverse-phase
chromatographies, and adsorption chromatographies (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et ai, Cold Spring Harbor
10 Laboratory Press, 1996). These chromatographies can be carried out using liquid phase
chromatographies such as HPLC and FPLC. Examples of the affinity chromatography columns
include protein A columns and protein G columns. Examples of the proteins A columns include
Hyper D, POROS, and Sepharose F. F. (Pharmacia).
[0062]
1 5 An antibody can be modified freely and peptide portions deleted by treating the
antibody with an appropriate protein modifying enzyme before or after antibody purification.
Such protein modifying enzymes include, for example, trypsins, chymotrypsins, lysyl
endopeptidases, protein kinases, and glucosidases.
[0063]
20 Antibodies that bind to Mpl can be prepared by methods known to those skilled in the
art.
For example, monoclonal antibody-producing hybridomas can be essentially generated
by known technologies as follows: immunizing animals with Mpl proteins or Mpl-expressing
cells as sensitized antigens using conventional immunological methods; fusing the obtained
25 immunocytes with known parental cells by conventional cell fusion methods; and screening for
monoclonal antibody-producing cells by conventional methods.
Specifically, monoclonal antibodies can be prepared by the method below.
First, Mpl protein, which is used as a sensitized antigen for preparing antibodies, is
prepared by expressing the Mpl gene/amino acid sequence (GenBank accession number:
30 NM_005373). More specifically, the gene sequence encoding Mpl is inserted into a known
expression vector, which is then transfected into an appropriate host cell. The subject human
Mpl protein is purified from the host cell or culture supernatant using known methods.
The purified Mpl protein is then used as a sensitized antigen. Alternatively, a partial
Mpl peptide may be used as a sensitized antigen. In this case, the partial peptide can also be
35 chemically synthesized based on the amino acid sequence of human Mpl.
[0064]
33
JP 2003-415746
The epitopes of Mpl molecule that are recognized by an anti-Mpl antibody of the
present invention are not limited to a particular epitope, and may be any epitope on the Mpl
molecule. Thus, any fragment can be used as an antigen for preparing anti-Mpl antibodies of
the present invention, as long as the fragment comprises an epitope of the Mpl molecule.
5 [0065]
There is no limitation as to the type of mammalian species to be immunized with the
sensitized antigen. However, a mammal is preferably selected based on its compatibility with
the parental cell to be used in cell fusion. Generally, rodents (for example, mice, rats, and
hamsters), rabbits, and monkeys can be used.
10 [0066]
Animals can be immunized with a sensitized antigen by known methods such as a
routine method of injecting a sensitized antigen into a mammal intraperitoneally or
subcutaneously. Specifically, the sensitized antigen is diluted appropriately with
phosphate-buffered saline (PBS), physiological saline and such, and then suspended. An
15 adequate amount of a conventional adjuvant, for example, Freund's complete adjuvant, is mixed
with the suspension, as necessary. An emulsion is then prepared for administering to a mammal
several times over a 4- to 21 -day interval. An appropriate carrier may be used for the sensitized
antigen in immunization.
A mammal is immunized as described above. After a titer increase of target antibody
20 in the serum is confirmed, immunocytes are collected from the mammal and then subjected to
cell fusion. Spleen cells are the preferred immunocytes.
[0067]
Mammalian myeloma cells are used as the parental cells to be fused with the above
immunocytes. Preferable myeloma cells to be used include various known cell lines, for
25 example, P3 (P3x63Ag8.653) (Kearney JF, et aL, J. Immnol. (1979) 123: 1548-1550),
P3x63Ag8U.l (Yelton DE, et aL, Current Topics in Microbiology and Immunology (1978) 81 :
1-7), NS-1 (Kohler, G and Milstein, C. Eur. J. Immunol. (1976) 6: 511-519), MPC-11 (Margulies,
D. H. etaL, Cell (1976) 8: 405-415), SP2/0 (Shulman, M. etal, Nature (1978) 276: 269-270),
FO (deSt. Groth, S. F. et aL, J. Immunol. Methods (1980) 35: 1-21), S194 (Trowbridge, I. S., J.
30 Exp. Med. (1978) 148: 313-323), and R210 (Galfre, G etaL 9 Nature (1979) 277: 131-133).
[0068]
Cell fusions between the immunocytes and the myeloma cells as described above can be
essentially carried out using known methods, for example, a method by Kohler and Milstein
(Kohler, G and Milstein, C, Methods Enzymol. (1981) 73: 3-46).
35 [0069]
More specifically, the above-described cell fusions are carried out, for example, in a
34
JP 2003-415746
conventional culture medium in the presence of a cell fusion-promoting agent. The
fusion-promoting agents include, for example, polyethylene glycol (PEG) and Sendai virus
(HVJ). If required, an auxiliary substance such as dimethyl sulfoxide may also be added to
improve fusion efficiency.
5 [0070]
The ratio of immunocytes to myeloma cells may be determined at one's own discretion,
preferably, for example, one myeloma cell for every one to ten immunocytes. Culture media to
be used for the above cell fusions include, for example, media that are suitable for the growth of
the above myeloma cell lines, such as RPMI 1 640 media and MEM media, and other
10 conventional culture media used for this type of cell culture. In addition, serum supplements
such as fetal calf serum (FCS) may also be used in combination.
[0071]
Cell fusion is carried out as follows. As described above, predetermined amounts of
immunocytes and myeloma cells are mixed well in the culture medium. PEG solution (for
15 example, mean molecular weight of about 1,000-6,000) pre-heated to 37°C is added to the cell
suspension typically at a concentration of 30% to 60% (w/v), and mixed to produce fused cells
(hybridomas). Then, an appropriate culture medium is successively added to the mixture, and
the sample is centrifuged to remove supernatant. This treatment is repeated several times to
remove the unwanted cell fusion-promoting agent and others that are unfavorable to hybridoma
20 growth.
[0072]
Screening of the resulting hybridomas can be carried out by culturing them in a
conventional selective medium, for example, hypoxanthine, aminopterin, and thymidine (HAT)
medium. Culturing in the above-descried HAT medium is continued for a period long enough
25 (typically, for several days to several weeks) to kill cells (non-fused cells) other than the desired
hybridomas. Then, hybridomas are screened for single-cell clones capable of producing the
target antibody by conventional limiting dilution methods.
[0073]
In addition to the method for preparing the above-descried hybridomas by immunizing
30 non-human animals with antigens, preferred human antibodies having binding activity to Mpl
can also be obtained by: sensitizing human lymphocytes with Mpl in vitro; and fusing the
sensitized lymphocytes with human myeloma cells capable of dividing permanently (see,
Japanese Patent Application Kokoku Publication No. (JP-B) HO 1-59878 (examined, approved
Japanese patent application published for opposition)). Alternatively, it is possible to obtain
35 human antibodies against Mpl from immortalized cells producing anti-Mpl antibodies. In this
method, the cells producing anti-Mpl antibodies are prepared by administering Mpl as an antigen
35 JP 2003-415746
to transgenic animals comprising a repertoire of the entire human antibody genes (see, WO
94/25585, WO 93/12227, WO 92/03918, and WO 94/02602).
The monoclonal antibody-producing hybridomas thus prepared can be passaged in a
conventional culture medium, and stored in liquid nitrogen over long periods of time.
5 [0074]
Monoclonal antibodies can be prepared from the above-described hybridomas by, for
example, a routine procedure of culturing the hybridomas and obtaining antibodies from the
culture supernatants. Alternatively, monoclonal antibodies can be prepared by injecting the
hybridomas into a compatible mammal; growing these hybridomas in the mammal; and
10 obtaining antibodies from the mammal's ascites. The former method is suitable for preparing
highly purified antibodies, while the latter is suitable for preparing antibodies on a large scale.
[0075]
Recombinant antibodies can also be prepared by: cloning an antibody gene from a
hybridoma; inserting the gene into an appropriate vector; introducing the vector into a host; and
1 5 producing the antibodies by using genetic recombination techniques (see, for example,
Vandamme, A. M. et al, Eur. J. Biochem. (1990) 192: 767-775).
[0076]
Specifically, an mRNA encoding the variable (V) region of anti-Mpl antibody is isolated
from hybridomas producing the anti-Mpl antibodies. For mRNA isolation, total RNAs are first
20 prepared by conventional methods such as guanidine ultracentrifugation methods (Chirgwin, J.
M. et aL, Biochemistry (1979) 18: 5294-5299), or acid guanidinium
thiocyanate-phenol-chloroform (AGPC) methods (Chomczynski, P. et a/., Anal. Biochem. (1987)
162: 156-159), and then the target mRNA is prepared using an mRNA Purification Kit
(Pharmacia) and such. Alternatively, the mRNA can be directly prepared using the QuickPrep
25 mRNA Purification Kit (Pharmacia).
[0077]
A cDNA of the antibody V region is synthesized from the resulting mRNA using reverse
transcriptase. cDNA synthesis is carried out using the AMV Reverse Transcriptase First-strand
cDNA Synthesis Kit (Seikagaku Co.), or such. Alternatively, cDNA can be synthesized and
30 amplified by the 5'-RACE method (Frohman, M. A. et al, Proc. Natl. Acad. Sci. USA (1988) 85:
8998-9002; Belyavsky, A. et aL, Nucleic Acids Res. (1989) 17: 2919-2932) using the 5'-Ampli
FINDER RACE Kit (Clontech) and PCR.
[0078]
Target DNA fragments are purified from the obtained PCR products and then ligated
35 with vector DNAs to prepare recombinant vectors. The vectors are introduced into E. coli and
such, and colonies are selected for preparing the recombinant vector of interest. The target
36
JP 2003-415746
DNA nucleotide sequence is then confirmed by conventional methods such as the
dideoxynucleotide chain termination method.
Once a DNA encoding the V region of target anti-Mpl antibody is obtained, the DNA is
inserted into an expression vector which comprises a DNA encoding the constant region (C
5 region) of a desired antibody.
The method for producing anti-Mpl antibodies to be used in the present invention
typically comprises the steps of: inserting an antibody gene into an expression vector, so that the
gene is expressed under the regulation of expression regulatory regions, such as enhancer and
promotor; and transforming host cells with the resulting vectors to express antibodies.
10 For expressing the antibody gene, polynucleotides encoding H chain and L chain,
respectively, are inserted into separate expression vectors and co-transfected into a host cell.
Alternatively, polynucleotides encoding both H chain and L chain are inserted into a single
expression vector and transfected into a host cell (see WO 94/11523).
[0079]
1 5 The term "agonistic activity" refers to an activity to induce changes in some biological
activities through signal transduction into cells and such, due to the binding of an antibody to a
receptor antigen. The biological activities include, for example, proliferation-promoting
activities, proliferation activities, viability activities, differentiation-inducing activities,
differentiation activities, transcriptional activities, membrane transport activities, binding
20 activities, proteolytic activities, phosphorylation/dephosphorylation activities,
oxidation/reduction activities, transfer activities, nucleolytic activities, dehydration activities,
cell death-inducing activities, and apoptosis-inducing activities, but is not limited thereto.
The term "agonistic activity against Mpl" typically refers to the activity of promoting
the differentiation of megakaryocytes or their parental hemopoietic stem cells into platelets, or
25 the activity of stimulating platelet proliferation.
Agonistic activity can be assayed by methods known to those skilled in the art. The
agonistic activity may be determined using the original activity or a different activity as an
indicator.
For example, agonistic activity can be determined by a method using cell growth as an
30 indicator as described in Examples. More specifically, an antibody whose agonistic activity is
to be determined is added to cells which proliferate in an agonist-dependent manner, followed by
incubation of the cells. Then, a reagent such as WST-8, which shows a coloring reaction at
specific wavelengths depending on the viable cell count, is added to the culture and absorbance
is measured. The agonistic activity can be determined using the measured absorbance as an
35 indicator.
[0080]
37
JP 2003-415746
Cells that proliferate in an agonist-dependent manner can also be prepared by methods
known to those skilled in the art. For example, when the antigen is a receptor capable of
transducing cell growth signals, cells expressing the receptor may be used. Alternatively, when
the antigen is a receptor that cannot transduce signals, a chimeric receptor consisting of the
5 intracellular domain of a receptor that transduces cell growth signals and the extracellular
domain of a receptor that does not transduce cell growth signals can be prepared for cellular
expression. Receptors that transduce cell growth signals include, for example, G-CSF receptors,
mpl, neu, GM-CSF receptors, EPO receptors, c-kit, and FLT-3. Cells that can be used to
express a receptor include, for example, BaF3, NFS 60, FDCP-1, FDCP-2, CTLL-2, DA-1, and
10 KT-3.
[0081]
There is no limitation as to the type of detection indicators to be used for determining
agonistic activity, as long as the indicator can monitor quantitative and/or qualitative changes.
For example, it is possible to use cell-free assay indicators, cell-based assay indicators,
15 tissue-based assay indicators, and in vivo assay indicators. Indicators that can be used in
cell-free assays include enzymatic reactions, quantitative and/or qualitative changes in proteins,
DNAs, or RNAs. Such enzymatic reactions include, for example, amino acid transfers, sugar
transfers, dehydrations, dehydrogenations, and substrate cleavages. Alternatively, protein
phosphorylations, dephosphorylations, dimerizations, multimerizations, hydrolyses, dissociations
20 and such; DNA or RNA amplifications, cleavages, and extensions can be used as the indicator in
cell-free assays. For example, protein phosphorylations downstream of a signal transduction
pathway may be used as a detection indicator. Alterations in cell phenotype, for example,
quantitative and/or qualitative alterations in products, alterations in growth activity, alterations in
cell number, morphological alterations, or alterations in cellular properties, can be used as the
25 indicator in cell-based assays. The products include, for example, secretory proteins, surface
antigens, intracellular proteins, and mRNAs. The morphological alterations include, for
example, alterations in dendrite formation and/or dendrite number, alteration in cell flatness,
alteration in cell elongation/axial ratio, alterations in cell size, alterations in intracellular
structure, heterogeneity/homogeneity of cell populations, and alterations in cell density. Such
30 morphological alterations can be observed under a microscope. Cellular properties to be used
as the indicator include anchor dependency, cytokine-dependent response, hormone dependency,
drug resistance, cell motility, cell migration activity, pulsatory activity, and alteration in
intracellular substances. Cell motility includes cell infiltration activity and cell migration
activity. The alterations in intracellular substances include, for example, alterations in enzyme
35 activity, mRNA levels, levels of intracellular signaling molecules such as Ca and cAMP, and
intracellular protein levels. When a cell membrane receptor is used, alterations in the cell
38 JP 2003-415746
proliferating activity induced by receptor stimulation can be used as the indicator. The
indicators to be used in tissue-based assays include functional alterations adequate for the subject
tissue. In in vivo assays, alterations in tissue weight, alterations in the blood system (for
example, alterations in blood cell counts, protein contents, or enzyme activities), alterations in
5 electrolyte levels, and alterations in the circulating system (for example, alterations in blood
pressure or heart rate).
[0082]
The methods for measuring such detection indices are not particularly limited. For
example, absorbance, luminescence, color development, fluorescence, radioactivity, fluorescence
1 0 polarization, surface plasmon resonance signal, time-resolved fluorescence, mass, absorption
spectrum, light scattering, and fluorescence resonance energy transfer may be used. These
measurement methods are known to those skilled in the art and may be selected appropriately
depending on the purpose. For example, absorption spectra can be obtained by using a
conventional photometer, plate reader, or such; luminescence can be measured with a
15 luminometer or such; and fluorescence can be measured with a fluorometer or such. Mass can
be determined with a mass spectrometer. Radioactivity can be determined with a device such
as a gamma counter depending on the type of radiation. Fluorescence polarization can be
measured with BEACON (TaKaRa). Surface plasmon resonance signals can be obtained with
BIACORE. Time-resolved fluorescence, fluorescence resonance energy transfer, or such can
20 be measured with ARVO or such. Furthermore, a flow cytometer can also be used for
measuring. It is possible to use one of the above methods to measure two or more different
types of detection indices. A greater number of detection indices may also be examined by
using two or more measurement methods simultaneously and/or consecutively. For example,
fluorescence and fluorescence resonance energy transfer can be measured at the same time with a
25 fluorometer.
[0083]
The present invention provides pharmaceutical compositions comprising antibodies of
this invention. The pharmaceutical compositions comprising antibodies of the present
invention are useful for treating and/or preventing thrombocytopenia and such. Time required
30 for the platelet count to recover to the normal level can be shortened by administering an
antibody of the present invention after donation of platelet components. The amount of platelet
components at the time of blood collection can be increased by pre-administering an antibody of
the present invention.
[0084]
35 When used as pharmaceutical compositions, the antibodies of the present invention can
be formulated by methods known to those skilled in the art. For example, the antibodies can be
39 JP 2003-415746
administered parenterally by injection of a sterile solution or suspension in water or other
pharmaceutically acceptable solvents. For example, the antibodies can be formulated by
appropriately combining with pharmaceutically-acceptable carriers or solvents, specifically,
sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants,
5 stabilizers, flavoring agents, excipients, vehicles, preservatives, binding agents, and such, and
mixing at a unit dosage and form required by accepted pharmaceutical implementations. In
such formulations, the amount of the thus obtained active ingredient should be within the
required range.
[0085]
10 A sterile composition to be injected can be formulated using a vehicle such as distilled
water used for injection, according to standard protocols.
[0086]
Aqueous solutions used for injections include, for example, physiological saline and
isotonic solutions comprising glucose or other adjunctive agents such as D-sorbitol, D-mannose,
15 D-mannitol, and sodium chloride. They may also be combined with an appropriate solubilizing
agent such as alcohol, specifically, ethanol, polyalcohol such as propylene glycol or polyethylene
glycol, or non-ionic detergent such as polysorbate 80 or HCO-50, as necessary.
[0087]
Oil solutions include sesame oils and soybean oils, and can be combined with
20 solubilizing agents such as benzyl benzoate or benzyl alcohol. Injection solutions may also be
formulated with buffers, for example, phosphate buffers or sodium acetate buffers; analgesics,
for example, procaine hydrochloride; stabilizers, for example, benzyl alcohol or phenol; or
anti-oxidants. The prepared injections are typically aliquoted into appropriate ampules.
[0088]
25 The administration is preferably carried out parenterally, specifically, by injection,
intranasal administration, intrapulmonary administration, percutaneous administration, or such.
Injections include, for example, intravenous injections, intramuscular injections, intraperitoneal
injections, and subcutaneous injections. The injection solutions can be also administered
systemically or locally.
30 [0089]
The administration methods can be selected properly according to the patient's age,
condition, and such. The applied dose of a pharmaceutical composition comprising an antibody
or polynucleotide encoding the antibody may be, for example, in the range of 0.0001 to 1,000
mg/kg body weight. Alternatively, the dosage may be, for example, in the range of 0.001 to
35 100,000 mg/kg body weight. However, the dosage is not restricted to the values described
above. The dosage and administration methods depend on the patient's weight, age, and
40
JP 2003-415746
condition, and are appropriately selected by those skilled in the art.
[Examples]
[0090]
The present invention is specifically illustrated below with reference to Examples, but it
5 is not to be construed as being limited thereto.
[Example 1 ] Preparation of anti-human Mpl antibodies
1 . 1 Establishment of Mpl-expressing BaF3 cell lines
BaF3 cell lines expressing the full-length Mpl gene were established to obtain cell lines
that proliferate in a TPO-dependent manner.
10 A full-length human Mpl cDNA (Palacios, R. et aL, Cell (1985) 41 : 727-734) (GenBank
accession NO. NM_005373) was amplified by PCR. The cDNA was cloned into a pCOS2
expression vector to construct pCOS2-hMplfull. The expression vector pCOS2 was
constructed by removing the DHFR gene expression region from pCHOI (Hirata, Y. et aL, FEBS
Letter (1994) 356: 244-248), where the expression region of the neomycin resistance gene
15 HEF-VH-gyl (Sato, K. etal, Mol Immunol. (1994) 31: 371-381) is inserted.
The cynomolgus monkey Mpl cDNA (SEQ ID NO : 1 64) was cloned from total RNA
extracted from the bone marrow cells of cynomolgus monkey, using a SMART RACE cDNA
Amplification Kit (Clontech). The resulting cynomolgus monkey cDNA was inserted into
pCOS2 to construct pCOS2-monkeyMplfull.
20 Then, the full-length mouse Mpl cDNA (GenBank accession NO. NMJ 1 0823) was
amplified by PCR, and inserted into pCOS2 to construct pCOS2-mouseMplfull.
Each vector (20 jag) prepared as described above was mixed with BaF3 cells (1 x 10 7
cells/mL) suspended in PBS in Gene Pulser cuvettes. This mixture was then pulsed at 0.33 kV
and 950 jiFD using a Gene Pulser II (Bio-Rad). The BaF3 cells introduced with the above
25 DNAs by electroporation were added to RPMI 1640 medium (Invitrogen) containing 1 ng/mL
mouse interleukin 3 (hereinafter abbreviated as mIL-3; Peprotech), 500 fag/mL Geneticin
(Invitrogen), and 10% FBS (Invitrogen), and selected to establish a human Mpl-expressing BaF3
cell line (hereinafter abbreviated as "BaF3 -human Mpl"), monkey Mpl-expressing BaF3 cell line
(hereinafter abbreviated as BaF3 -monkey Mpl), and mouse Mpl-expressing BaF3 cell line
30 (hereinafter abbreviated as "BaF3-mouse Mpl"). Following selection, these cells were cultured
and maintained in RPMI 1640 containing 1 ng/mL rhTPO (R&D) and 10% FBS.
[0091]
1 .2 Establishment of Mpl-expressing CHO cell lines
CHO cell lines expressing the full-length Mpl gene were established to obtain cell lines
35 to be used for assessing binding activity by flow cytometry.
First, the DHFR gene expression site from pCHOI was inserted into pCXN2 (Niwa, H.
JP 2003-415746
et al., Gene (1991) 108: 193-199) at the Hindlll site to prepare a pCXND3 expression vector.
The respective Mpl genes were amplified by PCR using pCOS2-hMplfull,
pCOS2-monkeyMplfull, and pCOS2-mouseMplfull as templates, and primers with a His-tag
sequence. The PCR products were cloned into pCXND3 to construct pCXND3-hMpl-His,
5 pCXND3 -monkey Mpl-His, and pCXND3 -mouse Mpl-His 5 respectively.
Vectors thus prepared (25 \xg each) were mixed with a PBS suspension of CHO-DG44
cells (1 x 10 7 cells/mL) in Gene Pulser cuvettes. The mixture was then pulsed at 1.5 kV and 25
jaFD using Gene Pulser II (Bio-Rad). The CHO cells introduced with these DNAs by
electroporation were added to CHO-S-SFMII medium (Invitrogen) containing 500 jag/mL
10 Geneticin and lx HT (Invitrogen). A human Mpl-expressing CHO cell line (hereinafter
abbreviated as "CHO-human Mpl"), monkey Mpl-expressing CHO cell line (hereinafter
abbreviated as "CHO-monkey Mpl"), and mouse Mpl-expressing CHO cell line (hereinafter
abbreviated as "CHO-mouse Mpl") were established through selection.
[0092]
15 1.3 Preparation of soluble human Mpl protein
To prepare soluble human Mpl protein, an expression system using insect Sf9 cells for
production and secretion of the protein was constructed as described below.
A DNA construct encoding the extracellular region of human Mpl (Gin 26 to Trp 491)
with a downstream FLAG tag was prepared. The construct was inserted into a pBACSurf-1
20 Transfer Plasmid (Novagen) between the Pstl and Smal sites to prepare pBACSurfl-hMpl-FLAG
Then, Sf9 cells were transformed with 4 jig of pBACSurfl-hMpl-FLAG using the Bac-N-Blue
Transfection Kit (Invitrogen). The culture supernatant was collected after three-day incubation.
Recombinant virus was isolated by plaque assays. The prepared virus stock was used to infect
Sf9 cells, and the culture supernatant was collected.
25 Soluble human Mpl protein was purified from the obtained culture supernatant as
described below. The culture supernatant was loaded onto a Q Sepharose Fast Flow
(Amersham Biosciences) for adsorption, and the adsorbed protein was then eluted with 50 mM
Na-phosphate buffer (pH7.2) containing 0.01% (v/v) Tween20 and 500 mM NaCl. After the
eluates were loaded onto a FLAG M2 -Agarose (Sigma- Aldrich) for adsorption, the protein
30 adsorbed was eluted with 100 mM glycine-HCl buffer (pH3.5) containing 0.01% (v/v) Tween20.
Immediately after elution, the fraction obtained was neutralized with 1 M Tris-Cl (pH8.0) and
the buffer was exchanged with PBS (-) and 0.01% (v/v) Tween20 using PD-10 columns
(Amersham Biosciences). The purified soluble Mpl protein was referred to as "shMpl-FLAG".
[0093]
35 1.4 Preparation of human Mpl-IgG Fc fusion protein
Human fusion protein Mpl-IgG Fc gene was prepared according to the method by
42
JP 2003-415746
Bennett et al (Bennett, B. D. et al. 9 J. Biol. Chem. (1991) 266: 23060-23067). A nucleotide
sequence encoding the extracellular region of human Mpl (Gin 26 to Tip 491) was linked to a
nucleotide sequence encoding the Fc region of human IgG-yl (a region downstream of Asp 216).
ABstEIl sequence (amino acids: Val-Thr) was attached to the junction as a fusion linker between
these two regions. A 19-amino acid signal peptide derived form human IgG H chain variable
region was used as the signal sequence. The resulting human fusion protein Mpl-IgG Fc gene
was cloned into pCXND3 to construct pCXND3-hMpl-Fc.
The vector thus prepared (25 (J.g) was mixed with a PBS suspension of CHO-DG44 cells
(1 x 10 7 cells/mL) in Gene Pulser cuvettes. The mixture was then pulsed at 1 .5 kV and 25 jaFD
using Gene Pulser II (Bio-Rad). The CHO cells introduced with the DNA by electroporation
were added to CHO-S-SFMII medium containing 500 jag/mL Geneticin and lx HT (Invitrogen).
shMPL-Fc-expressing CHO cell line (CHO-hMpl-Fc) was then established through selection.
Human Mpl-IgG Fc fusion protein was purified from the culture supernatant as
described below.
The culture supernatant was loaded onto a Q Sepharose Fast Flow (Amersham
Biosciences) for adsorption, and then the adsorbed protein were eluted with 50 mM
Na-phosphate buffer (pH7.6) containing 0.01% (v/v) Tween20 and 1 M NaCl. After the eluates
were loaded onto a HiTrap protein G HP column (Amersham Biosciences) for adsorption, the
adsorbed protein was eluted with 0.1 M glycine-HCl buffer (pH2.7) containing 150 mM NaCl
and 0.01% (v/v) Tween20. Immediately after elution, the obtained fraction was neutralized
with 1 M Tris-Cl (pH8.0) and the buffer was exchanged with PBS (-) and 0.01% (v/v) Tween20
using PD-10 columns (Amersham Biosciences). The purified soluble Mpl protein was referred
to as "hMpl-Fc".
[0094]
1.5 Immunization with shMpl-FLAG and hybridoma selection
MRL/Mp JUmmCrj -lpr/lpr mice (hereinafter abbreviated as "MRL/lpr mice"; purchased
from Charles River, Japan) were immunized; the primary immunization was carried out at eight
weeks of age. For every single mouse, an emulsion containing 100 jag of shMPL-FLAG
combined with Freund's complete adjuvant (H37 Ra; Beckton Dickinson), was administered
subcutaneously as the primary injection. As a booster injection, an emulsion containing
shMPL-FLAG (50 jag per mouse) combined with Freund's incomplete adjuvant (Beckton
Dickinson) was administered subcutaneously. Three mice which have been immunized six
times in total were subjected to a final injection of shMPL-FLAG (50 jig per mouse) through the
caudal vein. Cell fusion was achieved by mixing the mouse myeloma P3-X63Ag8Ul cells
(P3U1; purchased from ATCC) and mouse splenocytes using polyethylene glycol 1500 (Roche
Diagnostics). Hybridoma selection in HAT medium began the following day and culture
43
JP 2003-415746
supernatants were obtained. Screening was carried out by ELISA, using immunoplates
immobilized with shMpl-FLAG or hMpl-Fc and the assayed cell growth activity of BaF3-hMpl
as an index. Positive clones were isolated as single clones by limiting dilution and then
cultured in a large scale. The culture supernatants were collected.
5 [0095]
1.6 Analyses of anti-human Mpl antibodies
Antibody concentrations were determined by carrying out a mouse IgG sandwich
ELISA using goat anti-mouse IgG (gamma) (ZYMED) and alkaline phosphatase-goat
anti-mouse IgG (gamma) (ZYMED), generating a calibration curve by GraphPad Prism
10 (GraphPad Software; USA), and calculating the antibody concentrations from the calibration
curve. Commercially available antibodies of the same isotype were used as standards.
[0096]
Antibody isotypes were determined by antigen-dependent ELISA using isotype-specific
secondary antibodies. hMpl-Fc was diluted to 1 jag/mL with a coating buffer (0. 1 mM NaHCC>3,
15 pH9.6) containing 0.02% (w/v) NaN3, and then added to ELISA plates. The plates were
incubated overnight at 4°C for coating. The plates were blocked with a diluent buffer (50 mM
Tris-HCl (pH8.1) containing 1 mM MgCl 2 , 150 mM NaCl, 0.05% (v/v) Tween20, 0.02% (w/v)
NaN3, 1% (w/v) BSA). After the addition of hybridoma culture supernatants, the plates were
allowed to stand at room temperature for 1 hr. After washing with a rinse buffer (0.05% (v/v)
20 Tween20 in PBS), alkaline phosphatase-labeled isotype-specific secondary antibodies were
added to the plates. Then, the plates were allowed to stand at room temperature for 1 hr.
Color development was carried out using SIGMA 104 (Sigma- Aldrich) diluted to 1 mg/mL with
a substrate buffer (50 mM NaHC0 3 , pH9.8) containing 10 mM MgCl 2 , and absorbance was
measured at 405 nm using Benchmark Plus (Bio-Rad).
25 [0097]
The binding activities of an antibody to shMpl-FLAG and hMPL-Fc were determined
by ELISA. ELISA plates were coated with 1 |ig/mL of purified shMpl-FLAG or hMPL-Fc, and
blocked with a diluent buffer. Hybridoma culture supernatants were added to the plates, and the
plates were allowed to stand at room temperature for 1 hr. Then, alkaline phosphatase-labeled
30 anti -mouse IgG antibodies (Zymed) were added to the plates. Color development was similarly
carried out using the above method. Following a one-hour coloring reaction at room
temperature, absorbance was measured at 405 nm and EC50 values were computed using
GraphPad Prism.
[0098]
35 CHO-human Mpl cells and CHO-monkey Mpl cells were harvested, and suspended in
FACS Buffer (1% FBS/ PBS) to a final concentration of 1 x 10 6 cells/mL. The suspensions
44 JP 2003-415746
were aliquoted into Multiscreen (Millipore) at 1 00 jal/well, and the culture supernatants were
removed by centrifugation. Culture supernatants diluted to 5 |ug/mL were added to the plates
and incubated on ice for 30 min. The cells were washed once with FACS buffer, and incubated
on ice for 30 min following the addition of an FITC-labeled anti-mouse IgG antibody (Beckman
5 Coulter). After incubation, the mixture was centrifuged at 500 rpm for 1 min. The
supernatants were removed, and then the cells were suspended in 400 |uL of FACS buffer. The
samples were analyzed by flow cytometry using EPICS ELITE ESP (Beckman Coulter). An
analysis gate was set on the forward and side scatters of a histogram to include viable cell
populations.
10 [0099]
Agonistic activities of an antibody were evaluated using BaF3 -human Mpl and
BaF3 -monkey Mpl which proliferate in a TPO-dependent manner. Cells of each cell line were
suspended at 4 x 10 5 cells/ml in RPMI 1640/10% FBS (Invitrogen), and each suspension was
aliquoted into a 96-well plate at 60^1/well. A 40-jxL aliquot of rhTPO (R&D) and hybridoma
1 5 culture supernatants prepared at various concentrations was added into each well. The plates
were then incubated at 37°C under 5% C0 2 for 24 hr. A 10-jaL aliquot of the Cell Count
Reagent SF (Nacalai Tesque) was added into each well. After incubation for 2 hr, absorbance
was measured at 450 nm (and at 655 nm as a control) using a Benchmark Plus. EC 5 o values
were calculated using GraphPad Prism.
20 The above analysis yielded a total of 1 63 clones of mouse monoclonal antibodies that
bind to human Mpl.
[0100]
1.7 Purification of anti-human Mpl antibodies
Anti-human Mpl antibodies were purified from hybridoma culture supernatants as
25 described below.
After the culture supernatants were loaded onto HiTrap protein G HP columns
(Amersham Biosciences) for adsorption, the antibodies were eluted with 0.1 M glycine-HCl
(pH2.7). Immediately after elution, the fractions were neutralized with 1 M Tris-Cl (pH9.0),
dialyzed against PBS for one day, and the buffer was replaced.
30 [0101]
1.8 Determination of epitopes for the anti-human Mpl antibody VB22B
Since the anti-human Mpl antibody VB22B can be used for Western blotting, a
GST-fusion protein containing a partial sequence of human Mpl was constructed for VB22B
epitope analysis. MG1 (Gln26 to Trp491) and MG2 (Gln26 to Leu274) regions were each
35 amplified by PCR, and cloned into pGEX-4T-3 (Amersham Biosciences) to be expressed as GST
fusion proteins. The resulting plasmid DNAs were transformed into DH5ot to give
45 JP 2003-415746
transformants. A final concentration of 1 mM IPTG was added to the transformants in their
logarithmic growth phase to induce the expression of GST fusion proteins. The bacterial cells
were harvested after two hours of incubation. The cells were lysed by sonication. The lysates
were centrifuged in XL-80 Ultracentrifuge (Beckman, Rotor 70.1Ti) at 35,000 rpm for 30 min.
5 The culture supernatants were removed, and then the fusion proteins were purified using GST
Purification Modules (Amersham Biosciences). The samples were separated by
10%-SDS-PAGE, and then transferred onto a PVDF membrane. The membrane was Western
blotted with the murine antibody VB22B. VB22B was found to recognize both MG-1 and
MG-2, indicating that the VB22B epitope is located in the (Gln26 to Leu274) region.
10 [0102]
Then, GST fusion proteins containing the respective regions of human Mpl : MG3
(Gln26 to Alal89), MG4 (Gln26 to Prol06), MG5 (Gln26 to Glu259), and MG6 (Gln26 to
Gly245) were prepared and analyzed by Western blotting using the same procedure described
above. VB22B was found to recognize MG5 and MG6, but not MG3 and MG4. This
15 suggests that the VB22B epitope is located within the (Alal89 to Gly245) region. In addition,
GST was fused with MG7 (Gln26 to Ala23 1) and MG8 (Gln26 to Pro217) to prepare GST fusion
proteins. VB22B recognized MG7 but not MG8, suggesting that the VB22B epitope is located
in the (Gln2 1 7 to Ala23 1 ) region. Furthermore, GST fusion protein containing MG 1 0 (Gln2 1 3
to Ala231) was recognized by VB22B, suggesting that the VB22B epitope is located within the
20 limited region of 1 9 amino acids between Gln2 1 3 and Ala23 1 .
[0103]
1.9 Kinetic analyses of the antigen-antibody reaction for anti-human Mpl antibody VB22B
Since the anti-human Mpl antibody VB22B binds to soluble recombinant Mpl, kinetic
analyses of the antigen-antibody reaction between VB22B IgG and human Mpl-IgG Fc fusion
25 protein were carried out as described in Example 1 .4. The Sensor Chip CMS (Biacore) was
placed in Biacore 2000 (Biacore), and human Mpl-IgG Fc fusion protein was immobilized onto
the chip by amine-coupling methods. Then, 1.25 to 20 jag/mL of VB22B IgG solution was
prepared using HBS-EP Buffer (Biacore), and injected over the chip surface for 2 min to reveal
the binding region. Then, HBS-EP Buffer was injected over the chip surface for 2 min to reveal
30 the dissociation region. VB22B IgG bound to the human Mpl-IgG Fc fusion protein on the
sensor chip was removed by injecting 10 mM NaOH over the sensor chip for 15 sec, and the chip
was recovered. HBS-EP Buffer was used as the running buffer, and the flow rate was 20
(aL/min. Using the BIAevaluation Version 3.1 (Biacore) software, the reaction rate constant at
each concentration was calculated from the sensorgrams. The dissociation constant (KD) for
35 VB22B IgG was determined to be 1.67 ± 0.713 x 10" 9 M.
[0104]
46 JP 2003-415746
[Example 2] Preparation of single-chain anti-human Mpl antibodies
Among the prepared anti-human Mpl antibodies, 23 types of antibodies, which exhibit
higher binding activities and agonistic activities, were selected to construct expression systems
for single-chain antibodies using genetic engineering techniques. An exemplary method for
5 constructing a single-chain antibody derived from the anti-human Mpl antibody VB22B is
described below.
[0105]
2.1 Cloning of the anti-human Mpl antibody variable region
The variable region was amplified by RT-PCR using total RNA extracted from
10 hybridomas producing anti-human Mpl antibodies. Total RNA was extracted from 1 x 10 7
hybridoma cells using the RNeasy Plant Mini Kit (QIAGEN).
[0106]
A 5 '-terminal fragment of the gene was amplified from 1 jxg of total RNA by the
SMART RACE cDNA Amplification Kit (Clontech), using a synthetic oligonucleotide
15 MHC-IgG2b (SEQ ID NO: 166) complementary to mouse IgG2b constant region or a synthetic
oligonucleotide kappa (SEQ ID NO: 167) complementary to mouse k chain constant region.
Reverse transcription was carried out at 42 °C for 1.5 hr.
[0107]
The composition of the PCR reaction solution (50 jiL in total) is shown below.
1 Ox Advantage 2 PCR Buffer (Clontech) 5 ^iL
1 Ox Universal Primer A Mix (Clontech) 5 |liL
dNTPs (dATP, dGTP, dCTP, and dTTP) (Clontech) 0.2 mM
Advantage 2 Polymerase Mix (Clontech) 1 jiL
Reverse transcription product 2.5 |iL
Synthetic oligonucleotide, MHC-IgG2b or kappa 10 pmol
20 [0108]
The PCR reaction conditions were:
94°C (initial temperature) for 30 sec;
five cycles of 94°C for 5 sec and 72°C for 3 min;
five cycles of 94°C for 5 sec, 70°C for 10 sec, and 72°C for 3 min;
25 25 cycles of 94°C for 5 sec, 68°C for 10 sec, and 72°C for 3 min;
and final extension was at 72°C for 7 min.
[0109]
The PCR products were purified from agarose gel using the QIAquick Gel Extraction
Kit (QIAGEN), and cloned into a pGEM-T Easy Vector (Promega). The nucleotide sequence
30 was then determined using the ABI 3700 DN A Analyzer (Perkin Elmer).
47
JP 2003-415746
[0110]
The nucleotide sequence of cloned VB22B H chain variable region (hereinafter
abbreviated as "VB22B-VH") is shown in SEQ ID NO: 117, and its amino acid sequence is
shown in SEQ ID NO: 118. The nucleotide sequence of the L chain variable region (hereinafter
5 abbreviated as "VB22B- VL") is shown in SEQ ID NO: 119, and its amino acid sequence is
shown in SEQ ID NO: 120.
[0111]
2.2 Preparation of expression vectors for anti-human Mpl diabodies
The gene encoding VB22B single-chain Fv (hereinafter abbreviated as "VB22B
1 0 diabody") containing a five-amino acid linker sequence was constructed, by linking a nucleotide
sequence encoding a (Gly4Ser)i linker to the VB22B-VH-encoding gene at its 3' end and to the
VB22B-VL-encoding gene at its 5' end; both of which have been amplified by PCR.
[0112]
The VB22B-VH forward primer, 70115HF, (SEQ ID NO: 168) was designed to contain
1 5 an EcoW site. The VB22B-VH reverse primer, 33-11 5HR, (SEQ ID NO: 1 69) was designed to
hybridize to a DNA encoding the C terminus of VB22B-VH, and to have a nucleotide sequence
encoding the (Gly4Ser)i linker and a nucleotide sequence hybridizing to the DNA encoding the
N terminus of VB22B-VL. The VB22B-VL forward primer, 33-115LF, (SEQ ID NO: 170) was
designed to have a nucleotide sequence encoding the N terminus of VB22B-VL, a nucleotide
20 sequence encoding the (Gly4Ser)i linker, and a nucleotide sequence encoding the C terminus of
VB22B-VH. The VB22B-VL reverse primer, 33115LR, (SEQ ID NO: 171) was designed to
hybridize to a DNA encoding the C terminus of VB22B-VL and to have a nucleotide sequence
encoding a FLAG tag (Asp Tyr Lys Asp Asp Asp Asp Lys/SEQ ID NO: 172) and a Notl site.
[0113]
25 In the first round of PCR, two PCR products: one containing VB22B-VH and a linker
sequence, and the other containing VB22B-VL and the identical linker sequence, were
synthesized by the procedure described below.
The composition of the PCR reaction solution (50 jjL in total) is shown below.
1 Ox PCR Buffer (TaKaRa) 5 |xL
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 units
pGEM-T Easy vector comprising VB22B- VH or VB22B- VL gene 1 0 ng
Synthetic oligonucleotides, 70115HF and 33115HR, or 33-115LF 10 pmol
and33115LR
[0114]
30 The PCR reaction conditions were:
48 JP 2003-415746
94°C (initial temperature) for 30 sec;
five cycles of: 94°C for 15 sec and 72°C for 2 min;
five cycles of 94°C for 15 sec and 70°C for 2 min;
28 cycles of 94°C for 1 5 sec and 68°C for 2 min;
5 and final extension was at 72°C for 5 min.
[0115]
After the PCR products of about 400 bp were purified from agarose gel using the
QIAquick Gel Extraction Kit (QIAGEN), the second-round PCR was carried out using aliquots
of the respective PCR products according to the protocol described below.
10 The composition of the PCR reaction solution (50 ^iL in total) is shown below.
1 Ox PCR Buffer (TaKaRa) 5
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 unit
First-round PCR products (two types) 1 jjJL
Synthetic oligonucleotides, 70415HF and 33-115LR 10 pmol
[0116]
The reaction conditions were:
94°C (initial temperature) for 30 sec;
five cycles of 94°C for 15 sec and 72°C for 2 min;
1 5 five cycles of 94°C for 1 5 sec and 70°C for 2 min;
28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
[0117]
The PCR products of about 800 bp were purified from agarose gel using the QIAquick
20 Gel Extraction Kit (QIAGEN), and then digested with EcoRI and Notl (both from TaKaRa).
The resulting DNA fragments were purified using the QIAquick PCR Purification Kit
(QIAGEN), and then cloned into pCXND3 to prepare pCXND3-VB22B db.
[0118]
2.3 Preparation of expression vectors for anti-human Mpl antibody sc(Fv)2
25 To prepare expression plasmids for the modified antibody [sc(Fv)2] comprising two
units of H chain variable region and two units of L chain variable region derived from VB22B,
the above-described pCXND3-VB22B db was modified by PCR using the procedure shown
below. The process for constructing the sc(Fv)2 gene is illustrated in Fig. 1 .
[0119]
30 First, PCR method was carried out to amplify (a) the VB22B-VH-encoding gene in
which a nucleotide sequence encoding a 15-amino acid linker (Gly4Ser)3 was added to its 3' end;
49 JP 2003-415746
and (b) the VB22B-VL-encoding gene containing the identical linker nucleotide sequence added
to its 5' end. The desired construct was prepared by linking these amplified genes. Three new
primers were designed in this construction process. The VB22B-VH forward primer,
VB22B-fpvu, (primer A; SEQ ID NO: 173) was designed to have an EcoKL site at its 5' end and
5 to convert Gln22 and Leu23 of VB22B db into a Pvull site. The VB22B-VH reverse primer,
sc-rL15, (primer B; SEQ ID NO: 174) was designed to hybridize to a DNA encoding the C
terminus of VB22B-VH, and to have a nucleotide sequence encoding the (Gly4Ser)3 linker, as
well as a nucleotide sequence hybridizing to a DNA encoding the N terminus of VB22B-VL.
The VB22B-VL forward primer, sc-fL15, (primer C; SEQ ID NO: 175) was designed to have a
10 nucleotide sequence encoding the N terminus of VB22B-VL, a nucleotide sequence encoding the
(Gly4Ser) 3 linker, and a nucleotide sequence encoding the C terminus of VB22B-VH.
[0120]
In the first-round PCR, two PCR products: one comprising VB22B-VH and a linker
sequence, and the other comprising VB22B-VL and the identical linker sequence, were
1 5 synthesized by the procedure described below.
The composition of the PCR reaction solution (50 in total) is shown below.
1 Ox PCR Buffer (TaKaRa) 5 ^iL
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 units
pCXND3 -VB22B db 1 0 ng
Synthetic oligonucleotides, VB22B-fpvu, sc-rL15 or sc-fL15, and 10 pmol
33- 11 SLR (primer D)
[0121]
The reaction conditions were:
94°C (initial temperature) for 30 sec;
20 five cycles of 94°C for 15 sec and 72°C for 2 min;
five cycles of 94°C for 1 5 sec and 70°C for 2 min;
28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
[0122]
25 After the PCR products of about 400 bp were purified from agarose gel using the
QIAquick Gel Extraction Kit (QIAGEN), the second-round PCR was carried out using aliquots
of the respective PCR products according to the protocol described below.
The composition of the PCR reaction solution (50 jxL in total) is shown below.
1 Ox PCR Buffer (TaKaRa) 5
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
50 JP 2003-415746
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 units
First-round PCR product (two types) 1 jiL
Synthetic oligonucleotide, 70T15HF and 33115LR 10 pmol
[0123]
The reaction conditions were:
94°C (initial temperature) for 30 sec;
five cycles of 94°C for 15 sec and 72°C for 2 min;
5 five cycles of 94°C for 1 5 sec and 70°C for 2 min;
28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
[0124]
The PCR products of about 800 bp were purified from agarose gel using the QIAquick
10 Gel Extraction Kit (QIAGEN), and then digested with EcoRl and Notl (both from TaKaRa).
The resulting DNA fragments were purified using the QIAquick PCR Purification Kit
(QIAGEN), and then cloned into pBacPAK9 (Clontech) to construct pBacPAK9-scVB22B.
[0125]
A fragment to be inserted into the Pvull site of pBacPAK9-scVB22B was prepared.
15 Specifically, the fragment has a Pvull recognition site at both ends and a nucleotide sequence, in
which a gene encoding the VB22B-VH N-terminus is linked, via a (Gly4Ser) 3 linker-encoding
nucleotide sequence, to a gene encoding the amino acid sequence of an N terminus-deleted
VB22B-VH linked to VB22B-VL via the (Gly 4 Ser) 3 linker. Two primers were newly designed
to prepare the fragment by PCR. The forward primer for the fragment of interest, Fv2-f (primer
20 E; SEQ ID NO: 176), was designed to have a Pvull site at its 5' end and a VB22B-VH 5'-end
sequence. The reverse primer for the fragment of interest, Fv2-r (primer F; SEQ ID NO: 177),
was designed to hybridize to a DNA encoding the C terminus of VB22B-VL, and to have a Pvull
site, a nucleotide sequence encoding the (Gly 4 Ser)3 linker, and a nucleotide sequence hybridizing
to a DNA encoding the N terminus of VB22B-VH. PCR was carried out using
25 pBacPAK9-scVB22B as a template as described below.
[0126]
The composition of the PCR reaction solution (50 jiL in total) is shown below.
1 Ox PCR Buffer (TaKaRa) 5
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 units
pBacPAK9-scVB22B 1 0 ^ig
Synthetic oligonucleotide, Fv2-f and Fv2-r 10 pmol
[0127]
51
JP 2003-415746
The reaction conditions were:
94°C (initial temperature) for 30 sec;
five cycles of 94°C for 1 5 sec and 72°C for 2 min;
five cycles of 94°C for 1 5 sec and 70°C for 2 min;
5 28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
[0128]
The PCR products of about 800 bp were purified from agarose gel using the QIAquick
Gel Extraction Kit (QIAGEN), and then cloned into the pGEM-T Easy Vector (Promega).
1 0 After sequencing, the plasmid was digested with Pvull (TaKaRa), and the fragment of interest
was recovered. The recovered fragment was ligated to pBacPAK9-scVB22B pre-digested with
Pvull (TaKaRa) to construct pBacPAK9-VB22B sc(Fv)2. After the resulting vector was
digested with EcoKl and Notl (both from TaKaRa), the fragment of about 1,800 bp was purified
from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN). The fragment was then
15 cloned into a pCXND3 expression vector to construct pCXND3-VB22B sc(Fv)2.
[0129]
2.4 Expression of single-chain anti-human Mpl antibody in animal cells
A cell line stably expressing the single-chain antibody was prepared from CHO-DG44
cells as described below. Gene transfer was achieved by electroporation using a Gene Pulser II
20 (Bio-Rad). An expression vector (25 jig) and 0.75 mL of CHO-DG44 cells suspended in PBS
(1 x 10 7 cells/mL) were mixed. The resulting mixture was cooled on ice for 1 0 min, transferred
into a cuvette, and pulsed at 1 .5-kV and 25 jaFD. After a ten-minute restoration period at room
temperature, the electroporated cells were plated in CHO-S-SFMII medium (Invitrogen)
containing 500 jag/mL Geneticin (Invitrogen). CHO cell lines expressing the single-chain
25 antibody were established through selection. A cell line stably expressing VB22B sc(Fv)2 and
its culture supernatants were obtained by this method.
[0130]
The transient expression of the single-chain antibody was achieved using COS7 cells as
described below. An expression vector (10 jag) and 0.75 mL of CHO-DG44 cells suspended in
30 PBS (1 x 10 7 cells/mL) were mixed. The resulting mixture was cooled on ice for 1 0 min,
transferred into a cuvette, and then pulsed at 1 .5-kV and 25 jaFD. After a ten-minute restoration
period at room temperature, the electroporated cells were plated in DMEM/10% FBS medium
(Invitrogen). The cells were incubated overnight and then washed with PBS. CHO-S-SFMII
medium was added and the cells were cultured for about three days. The culture supernatants
35 for preparing the VB22B diabody were thus prepared.
[0131]
52
JP 2003-415746
2.5 Quantitation of single-chain anti-human Mpl antibodies in culture supernatants
The culture supernatant concentration of the single-chain anti-human Mpl antibody
transiently expressed in COS cells was determined using surface plasmon resonance. A sensor
chip CM5 (Biacore) was placed in Biacore 2000 (Biacore). ANTI-FLAG® M2 Monoclonal
5 Antibody (Sigma-Aldrich) was immobilized onto the chip. An appropriate concentration of
sample was injected over the chip surface at a flow rate of 5 mL/sec, and 50 mM diethylamine
was used to dissociate the bound antibody. Changes in the mass during sample injection were
recorded, and the sample concentration was calculated from the calibration curve prepared using
the mass changes of a standard sample. dbl2E10 (see WO 02/33073 and WO 02/33072) was
10 used as the diabody standard, and 12E10 sc(Fv)2 which has the same gene structure as that of
sc(Fv) 2 was used as the sc(Fv)2 standard.
[0132]
2.6 Purification of anti-human Mpl single-chain antibodies
The culture supernatants of VB22B diabody-expressing COS7 cells or CHO cells was
15 loaded onto an Anti-Flag M2 Affinity Gel (Sigma-Aldrich) column equilibrated with a 50 mM
Tris-HCl buffer (pH7.4) containing 150 mM NaCl and 0.05% Tween20. The absorbed
antibodies were eluted with 100 mM glycine-HCl (pH3.5). The fractions eluted were
immediately neutralized with 1 M Tris-HCl (pH8.0) 5 and loaded onto a HiLoad 26/60 Superdex
200 pg (Amersham Biosciences) column for gel filtration chromatography. PBS/0.01%
20 Tween20 was used in the gel filtration chromatography.
[0133]
VB22B sc(Fv)2 was purified from the culture supernatants of VB22B sc(Fv)2-expressing
COS7 cells or CHO cells under the same conditions used for purifying the diabodies. A
large-scale preparation of VB22B sc(Fv)2 was prepared by loading the CHO cell culture
25 supernatants onto a Macro-Prep Ceramic Hydroxyapatite Type I (Bio-Rad) column equilibrated
with a 20 mM phosphate buffer (pH6.8), and eluting the VB22B sc(Fv)2 in a stepwise manner
with 250 mM phosphate buffer (pH6.8). The eluted fraction was concentrated on an ultrafilter,
and then fractionated by gel filtration chromatography using a HiLoad 26/60 Superdex 200 pg
(Amersham Biosciences) column, and a fraction corresponding to the molecular weight range of
30 about 40 kD to 70 kD was obtained. The fraction was loaded onto an Anti-Flag M2 Affinity
Gel column equilibrated with a 50 mM Tris-HCl buffer (pH7.4) containing 150 mM NaCl and
0.05% Tween20. The absorbed antibody was eluted with 100 mM glycine-HCl (pH3.5). The
eluted fraction was immediately neutralized with 1 M Tris-HCl (pH8.0), and loaded onto a
HiLoad 26/60 Superdex 200 pg (Amersham Biosciences) column for gel filtration
35 chromatography. 20 mM acetate (pH6.0) containing 150 mM NaCl and 0.01% Tween80 was
used in the gel filtration chromatography.
JP 2003-415746
[0134]
2.7 Binding activity analyses of single-chain anti-human Mpl antibodies by flow cytometry
CHO-human Mpl, CHO-monkey Mpl, and CHO-mouse Mpl cells were recovered and
suspended in FACS buffer (1% FBS/PBS) to a final concentration of 1 x 10 6 cells/mL. Cell
5 suspensions were aliquoted at 100-jaL/well into the Multiscreen-HV Filter Plates (Millipore).
After centrifugation, the supernatant was removed. An appropriate concentration of diabody or
sc(Fv) 2 was added into each well and incubated on ice for 30 min. The cells were washed once
with 200 faL of FACS buffer, and incubated on ice for 30 min following the addition of 10 ng/mL
ANTI-FLAG® M2 Monoclonal Antibody (Sigma- Aldrich). The cells were then washed once
10 with 200 of FACS buffer, and a lOOx-diluted FITC-labeled anti-mouse IgG antibody
(Beckman Coulter) was added to the plate. The plate was incubated on ice for 30 min. After
centrifugation, the supernatant was removed. The cells were suspended in 400 \xL of FACS
Buffer, and then analyzed by flow cytometry using EPICS ELITE ESP (Beckman Coulter). An
analysis gate was set on the forward and side scatters of a histogram to include viable cell
15 populations.
[0135]
The binding activity of the purified VB22B sc(Fv) 2 to various Mpl molecules expressed
in CHO cells was determined (Fig. 2). VB22B sc(Fv) 2 was found to specifically bind to
CHO-human Mpl and CHO-monkey Mpl but not to the host cell CHO or CHO-mouse Mpl.
20 This binding characteristic of VB22B sc(Fv) 2 is comparable to those of VB22B IgQ indicating
that the antibody binding site remains unaltered by reduction of molecular weight.
[0136]
2.8 Analyses of TPO-like agonistic activity for single-chain anti-human Mpl antibodies
TPO-like agonistic activity was assessed using BaF3 -human Mpls or BaF3 -monkey
25 Mpls that proliferate in a TPO-dependent manner.
[0137]
Cells from each cell line were washed twice with RPMI 1640/1% FBS (fetal bovine
serum) (Invitrogen), and then suspended in RPMI 1640/10% FBS to a concentration of 4 x 10 5
cells/mL. Cell suspensions were aliquoted at 60-jo.L/well into a 96-well plate. Various
30 concentrations of rhTPO (R&D) and COS7 culture supernatants or purified samples were
prepared, and a 40-jjL aliquot was added into each well. The plates were then incubated at
37°C under 5% C0 2 for 24 hr. Immediately after a 10-jaL aliquot of WST-8 reagent (Cell
Count Reagent SF; Nacalai Tesque) was added into each well, absorbance was measured at 450
nm (and at 655 nm as a control) using Benchmark Plus. After two hours of incubation,
35 absorbance was again measured at 450 nm (and at 655 nm as a control). The WST-8 reagent
changes colors at 450 nm in a color reaction that reflects the viable cell count. The TPO-like
54 JP 2003-415746
agonistic activity was assessed using the change in absorbance during the two-hour incubation as
an index. EC50 values were computed using GraphPad Prism.
[0138]
TPO-like agonistic activity was assayed using the human leukemia cell line M-07e
5 (purchased from DSMZ) which proliferates TPO-dependently. M-07e cells were washed twice
with RPMI 1640/1% FBS, and then suspended in RPMI 1640/10% FBS to a concentration of 5 x
10 5 cells/mL. The resulting cell suspension was aliquoted at 50-|iL/well into a 96-well plate.
Various concentrations of rhTPO and COS7 culture supernatants or purified samples were
prepared, and a 50-jj.L aliquot was added into each well. The plates were then incubated at
10 37°C under 5% C0 2 for 48 hr. Immediately after a 10-^iL aliquot of WST-8 reagent (Cell
Count Reagent SF; Nacalai Tesque) was added to each well, absorbance of was measured at 450
nm (and at 655 nm as a control) using a Benchmark Plus. After four hours of incubation,
absorbance was again measured at 450 nm (and at 655 nm as a control). The TPO-like
agonistic activity was assayed using the change in absorbance during the four-hour incubation as
15 an index.
[0139]
Purified VB22B IgG, VB22B diabody, and VB22B sc(Fv) 2 were assayed for their
TPO-like agonistic activities using BaF3 -human Mpl, BaF3 -monkey Mpl, and M-07e. The
results are shown in Figures 3, 4, and 5, respectively. The presence of bivalent antigen-binding
20 domains in a single antibody molecule is essential for its agonistic activity. The distance and
angle between two antigen-binding domains can also be important factors (see WO 02/33073
and WO 02/33072). Similar results were obtained for the newly isolated anti-human Mpl
antibodies. Specifically, the agonistic activities of VB22B diabody and VB22B sc(Fv) 2 (EC50 =
61 pM and 27 pM in BaF -human Mpl, respectively) were higher than that of VB22B IgG (EC50
25 > 30 nM in BaF-human Mpl), and were equivalent to or higher than that of the
naturally-occurring human TPO ligand (EC50 = 76 pM in BaF-human Mpl). The VB22B
diabody activity was lower than that of VB22B sc(Fv)2. This suggests that the structure of a
single-chain antibody is greatly altered by its molecular shape and the length of the linker
sequence, which in turn changes the agonistic activity. Sixteen types of the single-chain
30 anti-human Mpl antibodies were obtained, each exhibiting a high agonistic activity. The amino
acid sequences of the H chain and L chain variable regions of the representative antibodies are
shown in Figures 6 and 7, respectively.
[0140]
2.9 Humanization of single-chain anti-human Mpl antibody
35 Antibody sequence data for the humanization of VB22B sc(Fv)2 were obtained from the
Kabat Database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/), and homology searches were carried
JP 2003-415746
out independently for the H chain variable region and the L chain variable region. As a result,
the H chain variable region was found to be highly homologous to DN13 (Smithson S. L. et aL,
Mol Immunol. (1999) 36: 113-124). The L chain variable region was found to be highly
homologous to ToP027 (Hougs L. et al 9 J. Immunol. (1999) 162: 224-237). Humanized
5 antibodies were prepared by inserting a complementarity-determining region (hereinafter
abbreviated as "CDR") into the framework regions (hereinafter abbreviated as "FR") of the
above antibodies.
[0141]
Specifically, synthetic oligo-DNAs of approximately 50 nucleotides in length were
1 0 designed as to make 20 of these nucleotides available for hybridization, and the synthetic
oligo-DNAs were assembled by PCR to prepare genes that encode the respective variable
regions. Using the resulting genes, sc(Fv)2 was similarly prepared by the method described in
Example 2.3. The respective DNAs were cloned into a pCXND3 expression vector to construct
expression vectors pCXND3-hVB22B sc(Fv)2. The nucleotide sequence and the amino acid
15 sequence of hVB22B sc(Fv) 2 in plasmid are shown in SEQ ID NO:l and SEQ ID NO: 2.
[0142]
[Example 3] Preparation of anti-Mpl diabodies by the AGS method
Anti-Mpl diabodies having agonistic activity were prepared by an Autocrine Growth
Selection (AGS) method (see, WO 03/91424).
20 [0143]
3 . 1 Construction of a retrovirus library
Spleens were isolated from MRL/lpr mice immunized with shMPL-Flag by the method
described in Example 1.5, and homogenized in TRIZOL Reagent (Invitrogen) using a Dounce
homogenizer. After chloroform addition, the homogenized sample was shaken vigorously, the
25 aqueous phase was removed and total RNA was extracted by isopropanol precipitation. mRNA
was purified using a PolyATract System 1000 (Promega). Reverse transcription of 2.5 \ig
mRNA was carried out at 42°C for 50 min using the Superscript First strand synthesis system for
RT-PCR (Invitrogen) and the included oligo-dT primers to prepare cDNA.
[0144]
30 The composition of the PCR reaction solution (250 jj,L) is shown below.
lOx KOD Plus Buffer (Toyobo) 25
2 mM dNTPs (dATP, dGTP, dCTP, and dTTP) (Toyobo) 25 yiL
2.5 mM MgS0 4 (Toyobo) 10 ^iL
KOD Plus (Toyobo) 7.5 \xL
Reverse transcription products 25 \xL
Mixed primers complementary to H chain or L chain variable region 500 pmol
JP 2003-415746
[0145]
The reaction conditions were:
98°C (initial temperature) for 3 min;
32 cycles of 98°C for 20 sec, 58°C for 20 sec, and 72°C for 30 sec;
5 and final extension was at 72°C for 6 min.
[0146]
The H chain primer mix contained HS1 to HS19 (SEQ ID NOs: 178 to 196) and HA1 to
HA4 (SEQ ID NOs: 197 to 200), which were mixed at the indicated ratios next to the sequence
names in Table 1 . The L chain primer mix contained LS 1 to LS 1 7 (SEQ ID NOs: 20 1 to 2 1 7),
10 LSlambda (SEQ ID NO: 218), LAI to LA5 (SEQ ID NOs: 219 to 222), and LAlambda (SEQ ID
NO: 223). The respective PCR products were purified from agarose gel using the QIAquick
Gel Extraction Kit (QIAGEN). The H chain and L chain variable regions were linked via the
(Gly 4 Ser)! linker sequence by PCR using sc-S (SEQ ID NO: 224) and sc-AS (SEQ ID NO: 225)
as described below.
15 [0147]
The composition of the PCR reaction solution (100 jaL in total) is shown below.
1 Ox KOD Plus Buffer (Toyobo) 1 0 jaL
2 mM dNTPs (dATP, dGTP, dCTP, and dTTP) (Toyobo) 1 0 jaL
2.5 mM MgS0 4 (Toyobo) 4 |aL
KOD Plus (Toyobo) 2 ^iL
Fragment of H chain variable region 4 jiL
Fragment of L chain variable region 4 nL
The first-round PCR conditions were:
94°C (initial temperature) for 3 min; and
seven cycles of 94°C for 1 min and 63 °C for 4 min.
20 Then, sc-S and sc-AS (25 pmol each) were added to the first-round products.
[0148]
The second-round PCR conditions were:
30 cycles of 94°C for 30 sec, 55°C for 2 min, and 72°C for 2 min;
and final extension was at 72°C for 6 min.
25 [0149]
The resulting product with an Sfil restriction site at both ends was purified using the
QIAquick PCR Purification Kit (QIAGEN), and incubated with the Sfil restriction enzyme
(TaKaRa) overnight at 50°C. The PCR product purified from agarose gel using the QIAquick
Gel Extraction Kit (QIAGEN) was inserted into the Sfil site of the viral vector
30 pMX/IL3ssGFPHis.
57
JP 2003-415746
[0150]
The resulting plasmid was constructed by inserting a GFP gene, which has an EcoRI site,
mouse IL-3 signal sequence and Sfil site at its 5' end; and an Sfil site, His tag sequence,
termination codon, and Not! site at its 3 ' end, between the EcoRI and Notl sites on the pMX viral
5 vector (Onishi, M. et al. 9 Mol. Cell. Biol. 18: 3871-3879). The plasmid was introduced into the
ElectroMAX DH10B Tl phage resistant cells (Invitrogen) by electroporation (settings: 2.5 kV,
25 jjJF, and 100Q) using a Gene Pulser II (Bio-Rad). The cells were plated onto an LB- Agar
plate containing 1 00 jig/mL ampicillin. After overnight incubation, 1 x 1 0 7 colonies were
obtained. Colonies were recovered from the plate and plasmids were then extracted using the
1 0 QIAGEN Plasmid Maxi Kit (QIAGEN).
[0151]
58 JP 2003-415746
[Table 1]
SEO
ID
NO *
1 78
I / u
(HS1 ( 4 - )
\noi \ *s / /
ftpppAttPPfifiPPATnnpnnAKfiTRMArcp , r r PPAfSf2Af2Tp
SEO
ID
NO •
1 7Q
( HS9 ( A \ j
p p p a np pfznp p a Tfi^rrzfi a firsT a p a o ptt* p a p p a ctp
SEO
TD
NO ■
1 SO
\ n o o \ o t j
f3PPP APPPPPPPATPPPPPAPPTPP A OPTO A APQ A QTP
SEO
NO *
181
1 O 1
( HQ. A (A) 1
<z p p p a op pon p p a Tncmc a <"2f2TP p a i? cvci raarapTP
SEO
TD
NO *
1 89
\ li o O \ i } J
SEO
TD
NO •
1
( MQfi foil
SEO
Tn
NO *
1 8-d
uLv.LAuv,v.uuv.v.A 1 ubLbLAub 1 L.LALu 1 oAAuLAb x V-
SEO
TD
NO •
nu .
1 8^
f UCQ / 9 ^ j
uL.V.LAVjLV.'jvjL.^A 1 bbLbbAbb 1 VjAAo O 1 oo X ooAAl v_
SEO
NO •
1 RR
1 o o
f ucQ / *Z \ j
uLLLAotLouLLAl ooCooAVo 1 oAWo I xoo xooAox V_
SEO
Tn
NO -
1 87
Vrio 1U \ zj
i ppppxppppppp'iT'Ppppr'APir'TPPiPcvpp'Ppp * r^fj^r^
J bLLLAbLLbbLLA 1 00L.00A00 X bLAuo ivoo 1 bbAb 1L
QUO
Tn
1 88
Vrio X 1 j
> bLLLAbLLbbLLA 1 ooCooAJvo 1 bLAMt I oo 1 ooAo l vU
day
Tn
XU
MO •
1 8Q
fuel o / o \
y utCL AbCLbbLLAl bbLbbAWj 1 bAAut 1 oA IbbAKTL
Tn
MO •
1 QD
fuel 1 / I ^
Vrio 1j llj
y bLLLAbLLbbLLA 1 ooC-ooAoo 1 bLAKL I TbTTbAbTC
SEO
Tn
MO -
1 Q1
Vrio X 4 \ Z )
y btt t Abv, L bb t L A i bbLbbAKb 1 KAAbb 1Tb xCbAbTC
Tn
MO •
fuel ^ i o \
y bt bbAbbbbbt bA 1 bbbbbAAb 1 bAAKbi IbAGbAGTC
oE,y
Tn
xu
f U C 1 £ / C \
^ ppppKPPPPPPPXHPPPPP>Pprnfnxpfnomni > xpupmcmp
/ bbbbAbbbbbCLATbGCbCAbbTTACTCTRAMbWGT
QUO
Tn
XU
MO -
1 OA
fuel n /
VHo X / t J .
O) / bCCCAbCCbbCCATbbCbCAGGTCCAACTVCAGCARCC
QUO
Tn
NO:
195
f uo 1 q i n
Vrio X o I U .
/ ; ^ bCbC AbCCbbbbAl GbbbbATG I bAACTTGGAAGTGTC
5>EQ
XLJ
NO:
196
(HS19 (0 .
7 ) ) GCCCAGCCGGCCATGGCGGAGGTGAAGGTCATCGAGTC
NO:
197
(HA1 (1) )
GGAGCCGCCGCCGCCCGAGGAAACGGTGACCGTGGT
ct?o
Tn
xu
NO:
198
(HA2 (1) )
GG AGCCGC C GC CGC C CG AGG AGAC TGTG AG AGTGGT
otto
XU
NO:
199
(HA3 (1) )
GG AG C CG CCGCCGCC CGC AG AG AC AGTG AC C AG AGT
oKQ
XU
NO:
200
(HA4 ( 1 ) )
GGAGCCGCCGCCGCCCGAGGAGACGGTGACTGAGGT
bEO
TO
xu
NO:
201
(LSI (1) )
GGCGGCGGCGGCTCCGAYATCCAGCTGACTCAGCC
SEO
Tn
J.U
NO:
202
(LS2 (2))
GGCGGCGGCGGCTCCGAYATTGTTCTCWCCCAGTC
SEO
Tn
NO: 203
(LS3 (5))
GGCGGCGG CGGC TC C GA YATTGTGMTMACTC AGT C
Tn
xu
NO:
204
(LS4 (3.5)) GGCGGCGGCGGCTCCGAYATTGTGYTRACACAGTC
Tn
xu
NO:
205
(LS5(4))
GGCGGCGGCGGCTCCGAYATTGTRATGACMCAGTC
Tn
xu
NO:
206
(LS6 (7))
GGCGGCGGCGGCTCCGAYATTMAGATRAMCCAGTC
QEO
Tn
xu
NO:
207
(LS7 (6) )
GGCGGCGGCGGCTCCGAYATTCAGATGAYDCAGTC
QUO
Tn
xu
NO:
208
(LS8 (1.5)) GGCGGCGGCGGCTCCGAYATYCAGATGACACAGAC
Tn
xu
NO:
209
(LS9 (2) )
GGCGGCGGCGGCTCCGAYATTGTTCTCAWCCAGTC
STPO
Ol*^
Tn
xu
NO:
210
(LS10 (3 .
5 ) ) GGCGGCGGCGGCTCCGAYATTGWGCTSACCCAATC
SEO
Tn
NO:
21 1
(LS11 (8)
) GGCGGCGGCGGCTCCGAYATTSTRATGACCCARTC
SEO
Tn
xu
NO:
212
(LS12 (8)
) GGCGGCGGCGGCTCCGAYRTTKTGATGACCCARAC
SEO
ID
NO:
213
(LS13 (6)
) GGCGGCGGCGGCTCCGAYATTGTGATGACBCAGKC
SEO
ID
NO:
214
(LS14 (2)
) GGCGGCGGCGGCTCCGAYATTGTGATAACYCAGGA
SEQ
ID
NO:
215
(LS15 (2)
) GGCGGCGGCGGCTCCGAYATTGTGATGACCCAGWT
SEQ
ID
NO:
216
(LS16 (1)
) GGCGGCGGCGGCTCCGAYATTGTGATGACACAACC
SEQ
ID
NO:
217
(LS17 (1)
) GGCGGCGGCGGCTCCGAYATTTTGCTGACTCAGTC
SEQ
ID
NO:
218
(LS lambda ( 1 ) ) GGCGGCGGCGGCTCCGATGCTGTTGTGACTCAGGAATC
SEQ
ID
NO:
219
(LAI (4))
GGAATTCGGCCCCCGAGGCCTTGATTTCCAGCTTGG
SEQ
ID
NO:
220
(LA2 (4))
GG AATTC GGCC C C CG AGG C CTTT ATTT C C AG CTTGG
SEQ
ID
NO:
221
(LA4 (4) )
GGAATTCGGCCCCCGAGGCCTTTATTTCCAACTTTG
SEQ
ID
NO:
222 i
(LAS (4))
GGAATTCGGCCCCCGAGGCCTTCAGCTCCAGCTTGG
SEQ
ID
NO:
223 (
( LAlambda ( 1 ) ) GGAATTCGGCCCCCGAGGCCCCTAGGACAGTC AGTTTGG
59 JP 2003-415746
[0152]
3.2 Establishment of autonomously replicating cell lines by the AGS method
The resulting library was transfected into a packaging cell, Pt-E, (Morita, S. et ai, Gene
therapy 7: 1063-1066) using FuGENE 6 (Roche Diagnostics). Specifically, Pt-E was plated
5 onto 6-cm dishes and cultured in DMEM/1 0% FBS (Invitrogen). A mixture of FuGENE 6 and
the library was added to the plate the following day. The culture medium was exchanged the
next day, and the culture supernatant was collected 24 hours after that. 1 0 jag/mL polybrene
(Hexadimethrine Bromide; Sigma) and 2 ng/mL mIL-3 were added to the culture supernatant
containing recombinant virus particles. The viral solution was used to infect the BaF-monkey
10 Mpl target cells. The cells were washed with PBS the following day, and suspended in RPMI
1640/10% FBS without mIL-3. The suspension was plated onto a 96-well plate at a cell density
of 1,000 cells/well. Autonomously replicating cell lines (AB317 and AB324) were obtained
after seven days of incubation. Genomic DNAs were extracted from these cells using a
DNeasy Tissue Kit (QIAGEN), and the antibody genes were amplified by PCR.
15 [0153]
The composition of the PCR reaction solution (50 ^iL in total) is shown below.
1 Ox LA Taq Buffer (TaKaRa) 5 jiL
2 mM dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 5 ^iL
2.5 mM MgCU (TaKaRa) 5 ^iL
TaKaRa LA Taq (TaKaRa) 0.5 fiL
Genomic DNA 0.5 jig
AGSdbSl (SEQ ID NO: 226) and AGSdbAl (SEQ ID NO: 227) 25 pmol
[0154]
The reaction conditions were:
94°C (initial temperature) for 1 min;
20 30 cycles of 94°C for 30 sec, 60°C for 30 sec, and 70°C for 1 min;
and final extension was at 72°C for 6 min.
[0155]
The nucleotide sequence and the amino acid sequence of the H chain of cloned AB3 17
are shown in SEQ ID NOs: 154 and 155. The nucleotide sequence and the amino acid
25 sequence of AB3 17 L chain are shown in SEQ ID NOs: 156 and 1 57. The nucleotide sequence
and the amino acid sequence of AB324 H chain are shown in SEQ ID NOs: 158 and 159. The
nucleotide sequence and the amino acid sequence of AB324 L chain are shown in SEQ ID NOs:
160 and 161.
[0156]
30 3.3 Activity assays of the diabodies obtained by AGS method
JP 2003-415746
Each of the anti-Mpl diabodies obtained above was inserted into the pCXND3
expression vector. The PCR primers used are a synthetic oligonucleotide complementary to the
5 ' end of the diabody and containing an EcoKL site, and a synthetic oligonucleotide
complementary to the nucleotide sequence of the 3 ' end of the diabody and containing a FLAG
5 tag and a Notl site. The PCR product thus obtained was inserted into pCXND3 between the
EcoRI and Notl sites. The diabody was expressed transiently in COS7 cells by the method
described in Example 2.4. The culture supernatant was removed and the activity of the diabody
was evaluated.
[0157]
1 0 The binding activities of the diabodies were assessed by flow cytometry using CHO
cells that express Mpl derived from various species (Fig. 8). AB3 1 7 was proven to bind to
CHO-mouse Mpl.
[0158]
The TPO-like agonistic activities of the diabodies were evaluated using BaF-human Mpl,
1 5 BaF-monkey Mpl, and BaF-mouse Mpl (Figures 9, 1 0, and 11). AB3 1 7 had the highest
agonistic activity against human, monkey, and mouse Mpl, whereas AB324 showed the highest
agonistic activity against human and monkey Mpl.
[0159]
This proves that anti-Mpl diabodies having high agonistic activity can be obtained by
20 the AGS method.
[Brief Description of the Drawings]
[0160]
[Fig. 1] Fig. 1 demonstrates the strategy for preparing single-chain antibody sc(Fv)2.
[Fig. 2] Fig. 2 illustrates the assessment of VB22B sc(Fv) 2 binding activity using an
25 Mpl-expressing CHO cell line. Purified VB22B sc(Fv)2 was used.
[Fig. 3] Fig. 3 illustrates the assessment of VB22B antibody agonistic activity using
BaF-human Mpl.
[Fig. 4] Fig. 4 illustrates the assessment of VB22B antibody agonistic activity using
BaF-monkey Mpl.
30 [Fig. 5] Fig. 5 illustrates the assessment of VB22B antibody agonistic activity using
M-07e.
[Fig. 6] Fig. 6 shows the amino acid sequences of anti-human Mpl antibodies (H chains)
that exhibit higher agonistic activities when converted into minibodies.
[Fig. 7] Fig. 7 shows the amino acid sequences of anti-human Mpl antibodies (L chains)
35 which exhibit higher agonistic activities when converted into minibodies.
[Fig. 8] Fig. 8 illustrates the binding activity assessment of AB317 diabody using
61
JP 2003-415746
Mpl-expressing CHO cells. Both VB22B diabody (solid line) and AB3 1 7 diabody (broken
line) were obtained from COS7 culture supernatants.
[Fig. 9] Fig. 9 illustrates the agnostic activity assessment of AB324 and AB317
diabodies using BaF -human Mpl.
5 [Fig. 1 0] Fig. 1 0 illustrates the agnostic activity assessment of AB324 and AB3 1 7
diabodies using BaF-monkey Mpl.
[Fig. 11] Fig. 11 illustrates the agnostic activity assessment of AB324 and AB317
diabodies using BaF-mouse Mpl.
62
JP 2003-415746
10
30
[Sequence Listing]
SEQUENCE LISTING
5 <110> CHUGAI SEIYAKU KABUSH I K I KAISHA
<120> anti-MpI antibody
<130> C1-A0320
<160> 227
<170> Patent In version 3. 1
15 <210> 1
<211> 1572
<212> DNA
<213> Mus musculus
20 <400> 1
atggaatggc ctttgatctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactcccag 60
gttcagctgc agcagtctgg acctgagctg gtgaagcctg gggcctcagt gaagatttcc 120
25 tgcaaggctt ctggctatgc attcactaac tcctggatga actgggtgaa gcagaggcct 180
ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 240
gggaaattca gggtcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 300
gatatcagca gcctgacatc tgaggactct gcggtctact tctgtgcaag aggctatgat 360
gattactcgt ttgcttactg gggccaaggg actctggtca ctgtctctgc aggtggtggt 420
35 ggttcgggtg gtggtggttc gggtggtggc ggatcggata ttgtgatgac tcaggctgca 480
63 JP 2003-415746
ccctctatac ctgtcactcc tggagagtca gtatccatct cctgtaggtc tagtaagagt 540
ctcctgcata gtaatggcaa cacttacttg tattggttcc tgcagaggcc aggccagtct 600
5 cctcaactcc tgatatatcg gatgtccaac cttgcctcag gagtcccaga taggttcagt 660
ggcagtgggt caggaactgc tttcacactg agaatcagta gagtggaggc tgaggatgtg 720
ggtgtttatt actgtatgca acatatagaa tatcctttta cgttcggatc ggggaccaag 780
10
ctggaaataa aaggaggtgg tggatcgggt ggtggtggtt cgggaggcgg tggatcgcag 840
gttcagctgc agcagtctgg acctgagctg gtgaagcctg gggcctcagt gaagatttcc 900
15 tgcaaggctt ctggctatgc attcactaac tcctggatga actgggtgaa gcagaggcct 960
ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 1020
gggaaattca gggtcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 1080
20
gatatcagca gcctgacatc tgaggactct gcggtctact tctgtgcaag aggctatgat 1140
gattactcgt ttgcttactg gggccaaggg actctggtca ctgtctctgc aggtggtggt 1200
25 ggttcgggtg gtggtggttc gggtggtggc ggatcggata ttgtgatgac tcaggctgca 1260
ccctctatac ctgtcactcc tggagagtca gtatccatct cctgtaggtc tagtaagagt 1320
ctcctgcata gtaatggcaa cacttacttg tattggttcc tgcagaggcc aggccagtct 1380
30
cctcaactcc tgatatatcg gatgtccaac cttgcctcag gagtcccaga taggttcagt 1440
ggcagtgggt caggaactgc tttcacactg agaatcagta gagtggaggc tgaggatgtg 1500
35 ggtgtttatt actgtatgca acatatagaa tatcctttta cgttcggatc ggggaccaag 1560
64 JP 2003-415746
ctggaaataa aa 1572
<210> 2
5 <211> 524
<212> PRT
<213> Mus musculus
<400> 2
10 Met Glu Trp Pro Leu Me Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
15 10 15
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
15
Pro Gly Ala Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe
35 40 45
Thr Asn Ser Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu
20 50 55 60
Glu Trp Me Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn
65 70 75 80
25 Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp I le Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
30
Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
115 120 125
Gin Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly
35 130 135 140
65
JP 2003-415746
Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Val Met Thr Gin Ala Ala
145 150 155 160
Pro Ser Me Pro Val Thr Pro Gly Glu Ser Val Ser He Ser Cys Arg
5 165 170 175
Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp
180 185 190
10 Phe Leu Gin Arg Pro Gly Gin Ser Pro Gin Leu Leu lie Tyr Arg Met
195 200 205
Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
210 215 220
15
Gly Thr Ala Phe Thr Leu Arg lie Ser Arg Val Glu Ala Glu Asp Val
225 230 235 240
Gly Val Tyr Tyr Cys Met Gin His Me Glu Tyr Pro Phe Thr Phe Gly
20 245 250 255
Ser Gly Thr Lys Leu Glu Me Lys Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
25 Gly Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Gin Gin Ser Gly Pro
275 280 285
Glu Leu Val Lys Pro Gly Ala Ser Val Lys lie Ser Cys Lys Ala Ser
290 295 300
Gly Tyr Ala Phe Thr Asn Ser Trp Met Asn Trp Val Lys Gin Arg Pro
305 310 315 320
Gly Lys Gly Leu Glu Trp Me Gly Arg Me Tyr Pro Gly Asp Gly Glu
35 325 330 335
66 JP 2003-415746
Thr lie Tyr Asn Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp
340 345 350
Lys Ser Ser Ser Thr Ala Tyr Met Asp Me Ser Ser Leu Thr Ser Glu
5 355 360 365
Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe
370 375 380
10 Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly
385 390 395 400
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Me Val Met
405 410 415
15
Thr Gin Ala Ala Pro Ser Me Pro Val Thr Pro Gly Glu Ser Val Ser
420 425 430
lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr
20 435 440 445
Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser Pro Gin Leu Leu
450 455 460
25 Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
465 470 475 480
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me Ser Arg Val Glu
485 490 495
30
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His Me Glu Tyr Pro
500 505 510
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
35 515 520
10
20
67 JP 2003-415746
<210> 3
<211> 5
<212> PRT
<213> Mus musculus
<400> 3
Ser Ser Trp Met Asn
1 5
<210> 4
<211> 17
<212> PRT
15 <213> Mus musculus
<400> 4
Arg Thr Tyr Pro 6 1 y Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
Gly
25 <210> 5
<211> 13
<212> PRT
<213> Mus musculus
30 <400> 5
Gly Trp Me Leu Ala Asp Gly Gly Tyr Ser Phe Ala Tyr
1 5 10
35 <210> 6
<211> 5
68
JP 2003-415746
<212> PRT
<213> Mus musculus
<400> 6
5 Ser Ser Trp Met Asn
1 5
<210> 7
10 <211> 17
<212> PRT
<213> Mus musculus
<400> 7
15 Arg Me Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
Gly
20
<210> 8
<211> 9
<212> PRT
25 <213> Mus musculus
<400> 8
Gly Tyr Ala Asp Tyr Ser Phe Ala Tyr
1 5
30
<210> 9
<211> 5
<212> PRT
35 <213> Mus musculus
69 JP 2003-415746
<400> 9
Ser Ser Trp Met Asn
1 5
5
<210> 10
<211> 17
<212> PRT
<213> Mus musculus
10
<400> 10
Arg Me Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
15 Gly
<210> 11
20 <211> 9
<212> PRT
<213> Mus musculus
<400> 1 1
25 Gly Phe Gly Asp Tyr Ser Phe Ala Tyr
1 5
<210> 12
30 <211> 5
<212> PRT
<213> Mus musculus
35
<400> 12
Ser Ser Trp Met Asn
1 5
70
JP 2003-415746
<210> 13
<211> 17
5 <212> PRT
<213> Mus musculus
<400> 1 3
Arg Me Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
10 1 5 10 15
<210> 14
<211> 9
<212> PRT
<213> Mus musculus
<400> 1 4
Gly Tyr Ala Asp Tyr Ser Phe Ala Tyr
1 5
Gly
25
<210>
15
<211>
5
<212>
PRT
<213>
Mus musculus
30
<400>
15
Arg Ser Trp Met Asn
5
35
<210> 16
71 JP 2003-415746
10
<211> 17
<212> PRT
<213> Mus musculus
<400> 1 6
Arg He Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
<210> 17
<211> 9
15 <212> PRT
<213> Mus musculus
<400> 1 7
Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
20 1 5
<210> 18
<211> 5
25 <212> PRT
<213> Mus musculus
<400> 1 8
Asn Ser Trp Met Asn
30 1 5
<210> 19
<211> 17
35 <212> PRT
<213> Mus musculus
72
JP 2003-415746
<400> 19
Arg He Tyr Pro Gly Asp Gly Glu Thr Asn Asn Asn Gly Lys Phe Lys
15 10 15
5
Gly
10 <210> 20
<211> 9
<212> PRT
<213> Mus musculus
15 <400> 20
Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr
1 5
20 <210> 21
<211> 5
<212> PRT
<213> Mus musculus
25 <400> 21
Asn Tyr Trp Val Asn
1 5
30 <210> 22
<211> 17
<212> PRT
<213> Mus musculus
35 <400> 22
Arg lie His Pro Ser Asp Ser Glu Thr His Cys Asn Gin Lys Phe Lys
73
JP 2003-415746
10
15
Arg
5
<210> 23
<211> 6
<212> PRT
10 <213> Mus musculus
15
<400> 23
Gly Gly Trp Phe Ala Tyr
1 5
<210> 24
<211> 5
<212> PRT
20 <213> Mus musculus
25
<400> 24
Ser Ser Trp Met Asn
1 5
<210> 25
<211> 17
<212> PRT
30 <213> Mus musculus
<400> 25
Arg lie Tyr Pro Gly Asp Gly Glu Thr Asn Asn Asn Gly Lys Phe Lys
1 5 10 15
35
Gly
74
JP 2003-415746
<210> 26
5 <211> 9
<212> PRT
<213> Mus musculus
<400> 26
10 Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr
1 5
<210> 27
15 <211> 5
<212> PRT
<213> Mus musculus
<400> 27
20 Thr Ser Trp Met Asn
1 5
<210> 28
25 <211> 17
<212> PRT
<213> Mus musculus
<400> 28
30 Arg Me Tyr Pro Gly Asp Gly Glu Ala Asn Tyr Asn Gly Lys Phe Lys
15 10 15
Gly
35
75
JP 2003-415746
10
15
20
25
<210> 29
<211> 9
<212> PRT
<213> Mus musculus
<400> 29
Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr
1 5
<210> 30
<211> 5
<212> PRT
<213> Mus musculus
<400> 30
Ser Ser Trp Met Asn
1 5
<210> 31
<211> 17
<21 2> PRT
<213> Mus musculus
<400> 31
Arg I le Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
30 Gly
<210> 32
35 <211> 9
<212> PRT
76
JP 2003-415746
<213> Mus musculus
<400> 32
Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr
5 1 5
<210> 33
<211> 5
10 <212> PRT
<213> Mus musculus
<400> 33
Arg Ser Trp Met Asn
15 1 5
<210> 34
<211> 17
20 <212> PRT
<213> Mus musculus
<400> 34
Arg He Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe Lys
25 1 5 10 15
Gly
30
35
<210> 35
<211> 9
<212> PRT
<213> Mus musculus
<400> 35
77
JP 2003-415746
Gly Asp Gly Asp Tyr Ser Phe Ala Tyr
1 5
5 <210> 36
<211> 5
<212> PRT
<213> Mus musculus
10 <400> 36
Asn Ser Trp Met Asn
1 5
15 <210> 37
<211> 17
<212> PRT
<213> Mus musculus .
20 <400> 37
Arg He Tyr Pro Gly Asp Gly Glu Thr He Tyr Asn Gly Lys Phe Arg
15 10 15
Val
25
<210> 38
<211> 9
30 <212> PRT
<213> Mus musculus
<400> 38
Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
35 1 5
78
JP 2003-415746
<210> 39
<211> 5
<212> PRT
5 <213> Mus musculus
<400> 39
Asp Tyr Trp Val Asn
1 5
10
<210> 40
<211> 17
<212> PRT
IS <213> Mus musculus
<400> 40
Arg He His Pro Tyr Asp Ser Glu Thr His Tyr Asn Gin Lys Phe Lys
1 5 10 15
20
Asn
25 <210> 41
<211> 6
<212> PRT
<213> Mus musculus
30 <400> 41
Gly Gly Trp Phe Ala Ser
1 5
35 <210> 42
<211> 5
79
JP 2003-415746
<212> PRT
<213> Mus musculus
<400> 42
5 Asp Tyr Trp Met Asn
1 5
<210> 43
10 <211> 17
<212> PRT
<213> Mus musculus
<400> 43
15 Arg lie His Pro Phe Asp Ser Glu Thr His Cys Ser Gin Lys Phe Lys
15 10 15
Asn
20
<210> 44
<211> 6
<212> PRT
25 <213> Mus musculus
<400> 44
Gly Gly Trp Phe Ala Tyr
1 5
30
<210> 45
<211> 5
<212> PRT
35 <213> Mus musculus
80
JP 2003-415746
<400> 45
Asn Ser Trp Met Asn
1 5
5
<210>
46
<211>
17
<212>
PRT
<213>
Mus musculus
10
<400> 46
Arg He Tyr Pro Gly Asp Gly Glu Thr lie Tyr Asn Gly Lys Phe Arg
15 Val
<210> 47
20 <211> 9
<212> PRT
<213> Mus musculus
<400> 47
25 Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
1 5
<210> 48
30 <211> 5
<212> PRT
<213> Mus musculus
5
10
15
35
<400> 48
Asn Ser Trp Met Asn
1 5
JP 2003-415746
<210> 49
<211> 17
5 <212> PRT
<213> Mus musculus
<400> 49
*
Arg lie Tyr Pro Gly Asp Gly Asp Thr lie Tyr Asn Gly Asn Phe Lys
10 1 5 10 15
<210> 50
<211> 9
<212> PRT
<213> Mus musculus
<400> 50
Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
1 5
Gly
25
<210>
51
<211>
5
<212>
PRT
<213>
Mus musculus
30
<400>
51
Ser Tyr Thr Met Ser
5
35
<210> 52
82
JP 2003-415746
<211> 17
<212> PRT
<213> Mus musculus
5 <400> 52
Thr Me Ser Ser Gly Ser Ser Thr lie Tyr Tyr Ala Asp Thr Val Lys
15 10 15
Gly
10
<210> 53
<211> 6
15 <212> PRT
<213> Mus musculus
<400> 53
Arg Trp Phe Leu Asp Cys
20 1 5
<210> 54
<211> 5
25 <212> PRT
<213> Mus musculus
<400> 54
Ser Ser Trp Met Asn
30 1 5
<210> 55
<211> 17
35 <212> PRT
<213> Mus musculus
83
JP 2003-415746
<400> 55
Arg lie Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
5
Gly
10 <210> 56
<211> 9
<212> PRT
<213> Mus musculus
15 <400> 56
Ala Arg Lys Thr Ser Trp Phe Ala Tyr
1 5
»
20 <210> 57
<211> 6
<212> PRT
<213> Mus musculus
25 <400> 57
Ser Asp Tyr Ala Trp Ser
1 5
30 <210> 58
<211> 16
<212> PRT
<213> Mus musculus
35 <400> 58
Tyr lie Thr Tyr Ser Gly Tyr Ser lie Tyr Asn Pro Ser Leu Lys Ser
84
JP 2003-415746
10 15
<210> 59
<211> 7
<212> PRT
<213> Mus musculus
<400> 59
10 Gly Tyr Asp Asn Met Asp Tyr
1 5
<210> 60
15 <211> 16
<212> PRT
<213> Mus musculus
<400> 60
20 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 61
25 <211> 7
<212> PRT
<213> Mus musculus
<400> 61
30 Arg Met Ser Asn Leu Ala Ser
1 5
35
<210> 62
<211> 9
<212> PRT
85
JP 2003-415746
<213> Mus musculus
<400> 62
Met Gin His Leu Glu Tyr Pro Phe Thr
1 5
<210> 63
<211> 16
10 <212> PRT
<213> Mus musculus
<400> 63
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 1 5 10 15
<210> 64
<211> 7
20 <212> PRT
<213> Mus musculus
<400> 64
Arg Met Ser Asn Leu Ala Ser
25 1 5
<210> 65
<211> 9
30 <212> PRT
<213> Mus musculus
<400> 65
Met Gin His Leu Glu Tyr Pro Tyr Thr
35 1 5
86
JP 2003-415746
10
20
30
<210> 66
<211> 16
<212> PRT
<213> Mus musculus
<400> 66
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 67
<211> 7
<212> PRT
15 <213> Mus musculus
<400> 67
Arg Met Ser Asn Leu Ala Ser
1 5
<210> 68
<211> 9
<212> PRT
25 <213> Mus musculus
<400> 68
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 69
<211> 16
<212> PRT
35 <213> Mus musculus
87
JP 2003-415746
<400> 69
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
1 5 10 15
<210> 70
<211> 7
<212> PRT
<213> Mus musculus
<400> 70
Arg Met Ser Asn Leu Ala Ser
1 5
<210> 71
<211> 9
<212> PRT
<213> Mus musculus
<400> 71
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 72
<211> 16
<212> PRT
<213> Mus musculus
<400> 72
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
35
<210> 73
88
JP 2003-415746
<211> 7
<212> PRT
<213> Mus musculus
5 <400> 73
Arg Met Ser Asn Leu Ala Ser
1 5
10 <210> 74
<211> 9
<212> PRT
<213> Mus musculus
15 <400> 74
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
20 <210> 75
<211> 16
<212> PRT
<213> Mus musculus
25 <400> 75
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
1 5 10 15
30 <210> 76
<211> 7
<212> PRT
<213> Mus musculus
35 <400> 76
Arg Met Ser Asn Leu Ala Ser
89 JP 2003-415746
<210> 77
<211> 9
<212> PRT
<213> Mus musculus
<400> 77
10 Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 78
15 <211> 16
<212> PRT
<213> Mus musculus
<400> 78
20 Arg Ser Ser Lys Ser Leu Leu Tyr Ser Asn Gly Asn Me Tyr Leu Tyr
15 10 15
<210> 79
25 <211> 7
<212> PRT
<213> Mus musculus
<400> 79
30 Arg Met Ser Asn Leu Ala Ser
1 5
35
<210> 80
<211> 9
<212> PRT
90
JP 2003-415746
<213> Mus musculus
<400> 80
Met Gin His Leu Glu Tyr Pro Tyr Thr
5 1 5
<210> 81
<211> 16
10 <212> PRT
<213> Mus musculus
<400> 81
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 1 5 10 15
<210> 82
<211> 7
20 <212> PRT
<213> Mus musculus
<400> 82
Arg Met Ser Asn Leu Ala Ser
25 1 5
<210> 83
<211> 9
30 <212> PRT
<213> Mus musculus
<400> 83
Met Gin His Leu Glu Tyr Pro Tyr Thr
35 1 5
91
JP 2003-415746
10
<210> 84
<211> 16
<212> PRT
<213> Mus musculus
<400> 84
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 85
<211> 7
<212> PRT
15 <213> Mus musculus
<400> 85
Arg Met Ser Asn Leu Ala Ser
1 5
<210> 86
<211> 9
<212> PRT
25 <213> Mus musculus
<400> 86
Met Gin His Val Glu Tyr Pro Tyr Thr
1 5
<210> 87
<211> 16
<212> PRT
35 <213> Mus musculus
92 JP 2003-415746
<400> 87
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
5
<210> 88
<211> 7
<212> PRT
<213> Mus musculus
10
<400> 88
Arg Met Ser Asn Leu Ala Ser
1 5
15
<210> 89
<211> 9
<212> PRT
<213> Mus musculus
20
<400> 89
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
25
<210> 90
<211> 16
<212> PRT
<213> Mus musculus
30
<400> 90
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
35
<210> 91
93
JP 2003-415746
<211> 7
<212> PRT
<213> Mus musculus
5 <400> 91
Arg Met Ser Asn Leu Ala Ser
1 5
10 <210> 92
<211> 9
<212> PRT
<213> Mus musculus
15 <400> 92
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
20 <210> 93
<211> 16
<212> PRT
<213> Mus musculus
25 <400> 93
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
30 <210> 94
<211> 7
<212> PRT
<213> Mus musculus
35 <400> 94
Arg Met Ser Asn Leu Ala Ser
94
JP 2003-415746
1 5
<210> 95
5 <211> 9
<212> PRT
<213> Mus musculus
<400> 95
10 Met Gin His Me Glu Tyr Pro Phe Thr
1 5
<210> 96
15 <211> 16
<212> PRT
<213> Mus musculus
<400> 96
20 Arg Ser Ser Lys Ser Leu Leu Tyr Ser Asn Gly Asn Thr Tyr Leu Tyr
■
15 10 15
<210> 97
25 <211> 7
<212> PRT
<213> Mus musculus
<400> 97
30 Arg Met Ser Asn Leu Ala Ser
1 5
<210> 98
35 <211> 9
<212> PRT
95
JP 2003-415746
<213> Mus musculus
<400> 98
Met Gin His Leu Glu Tyr Pro Tyr Thr
5 1 5
<210> 99
<211> 16
10 <212> PRT
<213> Mus musculus
<400> 99
Arg Ser Ser Lys Ser Leu Leu Tyr Ser Asn Gly Asn He Tyr Leu Tyr
15 1 5 10 15
<210> 100
<211> 7
20 <212> PRT
<213> Mus musculus
<400> 100
Arg Met Ser Asn Leu Ala Ser
25 1 5
<210> 101
<211> 9
30 <212> PRT
<213> Mus musculus
<400> 101
Met Gin His Leu Glu Tyr Pro Tyr Thr
35 1 5
96
JP 2003-415746
<210> 102
<211> 16
<212> PRT
5 <213> Mus musculus
<400> 102
Arg Ser Ser Lys Ser Leu Leu His Asn Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 103
<211> 7
<212> PRT
15 <213> Mus musculus
<400> 103
Arg Met Ser Asn Leu Ala Ser
1 5
<210> 104
<211> 9
<212> PRT
25 <213> Mus musculus
<400> 104
Met Gin His Me Glu Tyr Pro Phe Thr
1 5
<210> 105
<211> 16
<212> PRT
35 <213> Mus musculus
97
JP 2003-415746
<400> 1 05
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 106
<211> 7
<212> PRT
<213> Mus musculus
<400> 1 06
Arg Met Ser Asn Leu Ala Ser
1 5
<210> 107
<211> 9
<212> PRT
<213> Mus musculus
<400> 1 07
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 108
<211> 15
<212> PRT
<213> Mus musculus
<400> 1 08
Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gin
15 10 15
35
<210> 109
98
JP 2003-415746
<211> 7
<212> PRT
<213> Mus musculus
5 <400> 109
Gly Ala Ser Asn Val Giu Ser
1 5
10 <210> 110
<211> 9
<212> PRT
<213> Mus musculus
15 <400> 110
Gin Gin Ser Arg Lys Val Pro Trp Thr
1 5
20 <210> 111
<211> 11
<212> PRT
<213> Mus musculus
25 <400> 1 1 1
Lys Ala Ser Gin Asn Val Gly Asn Me Me Ala
1 5 10
30 <210> 112
<211> 7
<212> PRT
<213> Mus musculus
35 <400> 112
Leu Ala Ser Tyr Arg Tyr Ser
99
JP 2003-415746
<210> 113
<211> 9
<212> PRT
<213> Mus musculus
<400> 1 1 3
10 Gin Gin Tyr Ser Ser Ser Pro Leu Thr
1 5
<210> 114
15 <211> 12
<212> PRT
<213> Mus musculus
<400> 114
20 Ser Ala Ser Ser Ser Val Ser Ser Ser His Leu Tyr
1 5 10
<210> 115
25 <211> 7
<212> PRT
<213> Mus musculus
<400> 1 1 5
30 Ser Thr Ser Asn Leu Ala Ser
1 5
35
<210> 116
<211> 9
<212> PRT
100 JP 2003-415746
30
<213> Mus musculus
<400> 1 1 6
His Gin Trp Ser Ser Tyr Pro Trp Thr
5 1 5
<210> 117
<211> 411
10 <212> DNA
<213> Mus musculus
<220>
<221> CDS
15 <222> (1)..(411)
<223>
<400> 1 1 7
atg gaa tgg cct ttg ate ttt etc ttc etc ctg tea gga act gca ggt 48
20 Met Glu Trp Pro Leu Me Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
15 10 15
gtc cac tec cag gtt cag ctg cag cag tct gga cct gag ctg gtg aag 96
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
25 20 25 30
cct ggg gec tea gtg aag att tec tgc aag get tct ggc tat gca ttc 144
Pro Gly Ala Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Ala Phe
35 40 45
act aac tec tgg atg aac tgg gtg aag cag agg cct gga aag ggt ctt 192
Thr Asn Ser Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu
50 55 60
35 gag tgg att gga egg att tat cct gga gat gga gaa act ate tac aat 240
Glu Trp He Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn
101
JP 2003-415746
65
70
75
80
ggg aaa ttc agg gtc aag gcc aca ctg act gca gac aaa tec tec age
Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
5 85 90 95
288
10
aca gcc tac atg gat ate age age ctg aca tct gag gac tct gcg gtc 336
Thr Ala Tyr Met Asp lie Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
tac ttc tgt gca aga ggc tat gat gat tac teg ttt get tac tgg ggc 384
Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
115 120 125
15 caa ggg act ctg gtc act gtc tct gca 411
Gin Gly Thr Leu Val Thr Val Ser Ala
130 135
20 <210> 118
<211> 137
<212> PRT
<213> Mus musculus
25 <400> 118
Met Glu Trp Pro Leu Me Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
15 10 15
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
30 20 25 30
Pro Gly Ala Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe
35 40 45
35
Thr Asn Ser Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu
50 55 60
10
102 JP 2003-415746
Glu Trp Me Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn
65 70 75 80
5 Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp Me Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
115 120 125
Gin Gly Thr Leu Val Thr Val Ser Ala
15 130 135
<210> 119
<211> 396
20 <212> DNA
<213> Mus musculus
<220>
<221> CDS
25 <222> (1). . (396)
<223>
<400> 1 1 9
atg agg tgc eta get gag ttc ctg ggg ctg ctt gtg ttc tgg att cct 48
30 Met Arg Cys Leu Ala Glu Phe Leu Gly Leu Leu Val Phe Trp Me Pro
15 10 15
35
gga gec att ggg gat att gtg atg act cag get gca ccc tct ata cct
Gly Ala Me Gly Asp Me Val Met Thr Gin Ala Ala Pro Ser Me Pro
20 25 30
96
103 JP 2003-415746
gtc act cct gga gag tea gta tec ate tec tgt agg tct agt aag agt
Val Thr Pro Gly Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser
35 40 45
144
5 etc ctg cat agt aat ggc aac act tac ttg tat tgg ttc ctg cag agg
Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg
50 55 60
192
cca ggc cag tct cct caa etc ctg ata tat egg atg tec aac ctt gee 240
10 Pro Gly Gin Ser Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala
65 70 75 80
tea gga gtc cca gat agg ttc agt ggc agt ggg tea gga act get ttc
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe
15 85 90 95
288
20
aca ctg aga ate agt aga gtg gag get gag gat gtg ggt gtt tat tac
Thr Leu Arg Me Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
100 105 110
tgt atg caa cat ata gaa tat cct ttt acg ttc gga teg ggg acc aag
Cys Met Gin His Me Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys
115 120 125
336
384
25 ctg gaa ata aaa
Leu Glu Me Lys
130
396
30 <210> 120
<211> 132
<212> PRT
<213> Mus musculus
35 <400> 120
Met Arg Cys Leu Ala Glu Phe Leu Gly Leu Leu Val Phe Trp Me Pro
104 JP 2003-415746
10 15
Gly Ala Me Gly Asp Me Val Met Thr Gin Ala Ala Pro Ser Me Pro
20 25 30
Val Thr Pro Gly Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser
35 40 45
Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg
10 50 55 60
Pro Gly Gin Ser Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala
65 70 75 80
15 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe
85 90 95
Thr Leu Arg Me Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
100 105 110
20
Cys Met Gin His Me Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys
115 120 125
Leu Glu Me Lys
25 130
<210> 121
<211> 762
30 <212> DNA
<213> Mus musculus
<400> 121
atggaatggc ctttgatctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactcccag 60
35
gttcagctgc agcagtctgg acctgagctg gtgaagcctg gggcctcagt gaagatttcc 120
105 JP 2003-415746
tgcaaggctt ctggctatgc attcactaac tcctggatga actgggtgaa gcagaggcct 180
ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 240
gggaaattca gggtcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 300
gatatcagca gcctgacatc tgaggactct gcggtctact tctgtgcaag aggctatgat 360
10 gattactcgt ttgcttactg gggccaaggg actctggtca ctgtctctgc aggtggtggt 420
ggttcggata ttgtgatgac tcaggctgca ccctctatac ctgtcactcc tggagagtca 480
15
gtatccatct cctgtaggtc tagtaagagt ctcctgcata gtaatggcaa cacttacttg 540
tattggttcc tgcagaggcc aggccagtct cctcaactcc tgatatatcg gatgtccaac 600
cttgcctcag gagtcccaga taggttcagt ggcagtgggt caggaactgc tttcacactg 660
20 agaatcagta gagtggaggc tgaggatgtg ggtgtttatt actgtatgca acatatagaa 720
tatcctttta cgttcggatc ggggaccaag ctggaaataa aa 762
25 <210> 122
<211> 254
<212> PRT
<213> Mus musculus
30 <400> 122
Met Glu Trp Pro Leu Me Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
15 10 15
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
35 20 25 30
106 JP 2003-415746
Pro Gly Ala Ser
35
Thr Asn Ser Trp
5 50
Glu Trp I le Gly
65
Val Lys I le Ser
40
Met Asn Trp Val
55
Arg He Tyr Pro
70
Cys Lys Ala Ser
Lys Gin Arg Pro
60
Gly Asp Gly Glu
75
Gly Tyr Ala Phe
45
Gly Lys Gly Leu
Thr I le Tyr Asn
80
10 Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp lie Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
15
Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
115 120 125
Gin Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Asp Me
20 130 135 140
Val Met Thr Gin Ala Ala Pro Ser lie Pro Val Thr Pro Gly Glu Ser
145 150 155 160
25 Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly
165 170 175
Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser Pro Gin
180 185 190
30
Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg
1 95 200 205
Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me Ser Arg
35 210 215 220
107
JP 2003-415746
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His lie Glu
225 230 235 240
Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
5 245 250
<210> 123
<211> 635
10 <212> PRT
<213> Homo sapiens
<400> 123
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala
15 1 5 10 15
Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala
20 25 30
20 Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu
35 40 45
Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin
50 55 60
25
Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys Pro Leu Ser
65 70 75 80
Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro
30 85 90 95
Asp Gin Glu Glu Val Arg Leu Phe Phe Pro Leu His Leu Trp Val Lys
100 105 110
35 Asn Val Phe Leu Asn Gin Thr Arg Thr Gin Arg Val Leu Phe Val Asp
115 120 125
i
108 JP 2003-415746
Ser Val Gly Leu Pro Ala Pro Pro Ser Me He Lys Ala Met Gly Gly
130 135 140
5 Ser Gin Pro Gly Glu Leu Gin Me Ser Trp Glu Glu Pro Ala Pro Glu
145 150 155 160
e Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro
165 170 175
10
Lys Asn Ser Thr Gly Pro Thr Val Me Gin Leu Me Ala Thr Glu Thr
180 185 190
Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala Leu Asp Gin
15 195 200 205
Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly Pro Lys Gin
210 215 220
20 Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser
225 230 235 240
Cys Leu Me Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp Leu Gin Leu
245 250 255
25
Arg Ser Glu Pro Asp Gly Me Ser Leu Gly Gly Ser Trp Gly Ser Trp
260 265 270
Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val Ala Leu Gly
30 275 280 285
Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys Gin Trp Gin
290 295 300
35
Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His Ser Arg Ala
305 310 315 320
109
JP 2003-415746
Arg Cys Cys Pro Arg Asp Arg Tyr Pro Me Trp Glu Asn Cys Glu Glu
325 330 335
5 Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe Ser Arg Cys
340 345 350
His Phe Lys Ser Arg Asn Asp Ser He Me His Me Leu Val Glu Val
355 360 365
10
Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser Pro Phe Trp
370 375 380
e His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His Trp Arg Glu
15 385 390 395 400
Me Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro Ser Ser Trp
405 410 415
20 Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly Glu Gly His
420 425 430
25
Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr
435 440 445
Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu Arg Ala Arg
450 455 460
Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp Ser Asp Pro
30 465 470 475 480
Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser Leu Val Thr
485 490 495
35 Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu
500 505 510
110 JP 2003-415746
Leu Arg Trp Gin
515
5 Trp Pro Ser Leu
530
Asp Thr Ala Ala
545
10
Glu Glu Val Glu
Arg Thr Pro Leu
15 580
Arg Leu Gin Pro
595
20 Pro Met Ala Glu
610
Phe Pro Ala His Tyr Arg
520
Pro Asp Leu His Arg Val
535
Leu Ser Pro Pro Lys Ala
550
Pro Ser Leu Leu Glu Me
565 570
Pro Leu Cys Ser Ser Gin
585
Ser Cys Leu Gly Thr Met
600
Ser Gly Ser Cys Cys Thr
615
Arg Leu Arg His Ala Leu
525
Leu Gly Gin Tyr Leu Arg
540
Thr Val Ser Asp Thr Cys
555 560
Leu Pro Lys Ser Ser Glu
575
Ala Gin Met Asp Tyr Arg
590
Pro Leu Ser Val Cys Pro
605
Thr His Me Ala Asn His
620
Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
25
<210> 124
<211> 122
<212> PRT
30 <213> Mus musculus
<400> 1 24
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
35
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
Ill
JP 2003-415746
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
Gly Arg Thr Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
10 65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
15 Ala Arg Gly Trp Me Leu Ala Asp Gly Gly Tyr Ser Phe Ala Tyr Trp
100 105 110
Gly Gin Gly Thr Leu Val Thr Val Ser Ala
115 120
20
<210> 125
<211> 112
<212> PRT
25 <213> Mus musculus
<400> 1 25
Asp Me Val Met Thr Gin Ala Ala Pro Ser Me Pro Val Thr Pro Gly
15 10 15
30
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 35 40 45
112 JP 2003-415746
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
5 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Me Tyr Tyr Cys Met Gin His
85 90 95
10 Leu Glu Tyr Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu I le Lys
100 105 110
<210> 126
15 <211> 118
<212> PRT
<213> Mus musculus
<400> 126
20 Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30
25
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
30 50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
35 Me Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
113 JP 2003-415746
Ala Arg Gly Tyr Ala Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
5 Leu Val Thr Val Ser Ala
115
<210> 127
10 <211> 112
<212> PRT
<213> Mus musculus
<400> 127
15 Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
1 5 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
20
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
25 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
30 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
35
114 JP 2003-415746
<210> 128
<211> 118
<212> PRT
<213> Mus musculus
<400> 1 28
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
10 Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp He
35 40 45
15
Gly Arg I le Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr
20 65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
25 Ala Arg Gly Phe Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
30
<210> 129
<211> 112
<212> PRT
35 <213> Mus musculus
115 JP 2003-415746
<400> 1 29
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
5 Glu Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
10
Pro Gin Leu Leu lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Ala Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
15 65 70 75 80
Ser Arg Val Glu Thr Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
20 Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
<210> 130
25 <211> 118
<212> PRT
<213> Mus musculus
<400> 130
30 Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser
20 25 30
35
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
116 JP 2003-415746
35 40 45
Gly Arg I le Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
5
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
10 85 90 95
Ala Ser Gly Tyr Ala Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
15 Leu Val Thr Val Ser Ala
115
<210> 131
20 <211> 112
<212> PRT
<213> Mus musculus
<400> 131
25 Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
Glu Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
35 50 55 60
117 JP 2003-415746
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg lie
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
5 85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys
100 105 110
i
10
<210> 132
<211> 118
<212> PRT
<213> Mus musculus
15
<400> 132
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
20 Ser Val Lys I le Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Arg Ser
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp I le
35 40 45
25
Gly Arg Me Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
30 65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
35 Ala Ser Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
118 JP 2003-415746
Leu Val Thr Val Ser Ala
115
5
<210> 133
<211> 112
<212> PRT
<213> Mus musculus
10
<400> 1 33
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
1 5 10 15
15 Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
20
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
25 .65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
30 Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
<210>
35 <211>
<212>
134
118
PRT
119 JP 2003-415746
<213> Mus musculus
<400> 1 34
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
5 1 5 10 15
Ser Val Lys He Ser Cys Arg Ala Phe Gly Tyr Ala Phe Ser Asn Ser
20 25 30
10 Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp I le
35 40 45
Gly Arg He Tyr Pro Gly Asp Gly Glu Thr Asn Asn Asn Gly Lys Phe
50 55 60
15
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
20 85 90 95
Ala Arg Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
25 Leu Val Thr Val Ser Ala
115
<210> 135
30 <211> 112
<212> PRT
<213> Mus musculus
<400> 1 35
35 Asp He Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
120 JP 2003-415746
Glu Ser Val Ser He Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
5 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
10
Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Ala Phe Thr Leu Arg Me
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
15 85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
20
<210> 136
<211> 115
<212> PRT
<213> Mus musculus
25
<400> 136
Gin Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
15 10 15
30 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Val Asn Trp Val Lys Gin Arg Pro Gly Arg Gly Leu Glu Trp Me
35 40 45
35
Gly Arg Me His Pro Ser Asp Ser Glu Thr His Cys Asn Gin Lys Phe
121 JP 2003-41 5746
50 55 60
Lys Arg Lys Ala Thr Leu Thr Val Asn Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
5
He Gin Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Ser Gly Gly Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr
10 100 105 110
Val Ser Ala
115
15
<210> 137
<211> 112
<212> PRT
<213> Mus musculus
20
<400> 1 37
Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
25 Glu Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser
20 25 30
Asn Gly Asn Me Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
30
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
35 65 70 75 80
122
JP 2003-415746
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
5 100 105 110
<210> 138
<211> 118
10 <212> PRT
<213> Mus musculus
<400> 1 38
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 1 5 10 15
Ser Val Lys lie. Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30
20 Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp He
35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Asn Asn Asn Gly Lys Phe
50 55 60
25
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
30 85 90 95
Ala Arg Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
35 Leu Val Thr Val Ser Ala
115
123 JP 2003-415746
<210> 139
<211> 112
5 <212> PRT
<213> Mus musculus
<400> 1 39
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
10 1 5 10 15
G I u Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
15 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
20
Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Ala Phe Thr Leu Arg He
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
25 85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys
100 105 110
30
<210> 140
<211> 118
<212> PRT
<213> Mus musculus
35
<400> 1 40
124 JP 2003-415746
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Thr Ser
5 20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp I le
35 40 45
10 Gly Arg He Tyr Pro Gly Asp Gly Glu Ala Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Ser Ala Tyr
65 70 75 80
15
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
20 100 105 110
Leu Val Thr Val Ser Ala
115
25
<210> 141
<211> 112
<21 2> PRT
<213> Mus musculus
30
<400> 141
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
35 Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
125 JP 2003-415746
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Met 61 n Arg Pro Gly Gin Ser
35 40 45
5 Pro Gin Leu Leu I le Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
10
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Val Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys
15 100 105 110
<210> 142
<211> 118
20 <212> PRT
<213> Mus musculus
<400> 142
Gin Val Gin Leu Gin
25 1 5
Ser Val Lys I le Ser
20
30 Trp Met Asn Trp Val
35
Gly Arg I le Tyr Pro
50
35
Lys Gly Lys Ala Thr
Gin Ser Gly Pro Glu
10
Cys Lys Ala Ser Gly
25
Lys Gin Arg Pro Gly
40
Gly Asp Gly Glu Thr
55
Leu Thr Ala Asp Lys
Leu Val Lys Pro Gly Ala
15
Tyr Ala Phe Ser Ser Ser
30
Lys Gly Pro Glu Trp I le
45
Asn Tyr Asn Gly Lys Phe
60
Ser Ser Ser Thr Val Tyr
126 JP 2003-415746
65
70
75
80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
10
Leu Val Thr Val Ser Ala
115
<210> 143
<211> 112
15 <212> PRT
<213> Mus musculus
<400> 143
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
20 1 5 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
25 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
30
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
35 85 90 95
127 JP 2003-415746
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
5 <210> 144
<211> 118
<212> PRT
<213> Mus musculus
10 <400> 144
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Leu Asn Pro Gly Ala
15 10 15
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Arg Ser
15 20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
20 Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
25
Met Gin Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Asp Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
30 100 105 110
Leu Val Thr Val Ser Ala
115
35
<210> 145
128 JP 2003-415746
<211> 112
<212> PRT
<213> Mus musculus
5 <400> 145
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
Glu Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
10 20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
15 Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg lie
65 70 75 80
20
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu He Lys
25 100 105 110
<210> 146
<211> 115
30 <212> PRT
<213> Mus musculus
35
<400> 1 46
Gin Val Gin Leu Gin Gin Pro Gly Thr Glu Leu Val Arg Pro Gly Ala
15 10 15
129 JP 2003-415746
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Trp Val Asn Trp Val Lys Gin Arg Pro Gly Arg Gly Leu Glu Trp Me
5 35 40 45
Gly Arg Me His Pro Tyr Asp Ser Glu Thr His Tyr Asn Gin Lys Phe
50 55 60
10 Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Me Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
15
Ala Ser Gly Gly Trp Phe Ala Ser Trp Gly Gin Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
20 115
<210> 147
<211> 112
25 <212> PRT
<213> Mus musculus
<400> 147
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
30 1 5 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser
20 25 30
35 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
130 JP 2003-415746
Pro Gin Leu Leu lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
5 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Thr I le
65 70 75 80
Ser Ser Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
10
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
15 <210> 148
<211> 115
<212> PRT
<213> Mus musculus
20 <400> 148
Gin Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
25 20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Arg Gly Leu Glu Trp lie
35 40 45
30 Gly Arg Me His Pro Phe Asp Ser Glu Thr His Cys Ser Gin Lys Phe
50 55 60
Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
35
Me Gin Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
131
JP 2003-415746
85
90
95
Ser Ser Gly Gly Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
115
10 <210> 149
<211> 112
<212> PRT
<213> Mus musculus
15 <400> 149
Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Ser Val Thr Pro Gly
15 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser
20 20 25 30
Asn Gly Asn Me Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
25 Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Me
65 70 75 80
30
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
35 100 105 110
132 JP 2003-415746
<210> 150
<211> 118
<212> PRT
5 <213> Mus musculus
<400> 150
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
10
Ser Val Lys I le Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Ser
20 25 30
Trp Met Asn Trp Val Arg Gin Arg Pro Gly Lys Gly Leu Glu Trp lie
15 35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr lie Tyr Asn Gly Lys Phe
50 55 60
20 Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Me Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
25
Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
30 115
<210> 151
<211> 112
35 <212> PRT
<213> Mus musculus
133
JP 2003-415746
<400> 151
Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Asn
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
10 35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
15 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
20
Me Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
25 <210> 152
<211> 118
<212> PRT
<213> Mus musculus
30 <400> 152
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Ser
35 20 25 30
134 JP 2003-415746
Trp Met Asn Trp Val Asn Gin Arg Pro Gly Lys Gly Leu Glu Trp I le
35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Asp Thr Me Tyr Asn Gly Asn Phe
5 50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Me Ala Tyr
65 70 75 80
10 Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Thr Ser Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
15
Leu Val Thr Val Ser Ala
115
20 <210> 153
<211> 112
<212> PRT
<213> Mus musculus
25 <400> 153
Asp Me Val Met Thr Gin Ala Ala Pro Ser Leu Pro Val Thr Pro Gly
15 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
30 20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
35 Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
135
JP 2003-415746
10
30
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
5 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys
100 105 110
<210> 154
<211> 423
<212> DNA
15 <213> Mus musculus
<220>
<221> CDS
<222> (1). . (423)
20 <223>
<400> 1 54
atg gtt ctt gcc age tct acc acc age ate cac ace atg ctg etc ctg 48
Met Val Leu Ala Ser Ser Thr Thr Ser Me His Thr Met Leu Leu Leu
25 1 5 10 15
etc ctg atg ctg gcc cag ccg gcc atg gcg gaa gtg aag ctg gtg gag 96
Leu Leu Met Leu Ala Gin Pro Ala Met Ala Glu Val Lys Leu Val Glu
20 25 30
tct ggg gga ggc tta gtg aag cct gga ggg tec egg aaa etc tec tgt 144
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Arg Lys Leu Ser Cys
35 40 45
35 gca gcc tct gga ttc act ttc agt age tat acc atg tct tgg gtt cgc 192
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser Trp Val Arg
136 JP 2003-415746
10
35
50 55 60
cag act ccg gcg aag agg ctg gag tgg gtc gca acc att agt agt ggc 240
Gin Thr Pro Ala Lys Arg Leu Glu Trp Val Ala Thr lie Ser Ser Gly
5 65 70 75 80
agt agt acc ate tac tat gca gac aca gtg aag ggc cga ttc acc ate 288
Ser Ser Thr Me Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr lie
85 90 95
tec aga gac aat gec aag aac acc ctg ttc ctg caa atg acc agt eta 336
Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe Leu Gin Met Thr Ser Leu
100 105 110
15 agg tct gag gac aca gec atg tat tac tgt gca agg aga tgg ttt ctt 384
Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Trp Phe Leu
115 120 125
gac tgc tgg ggc caa ggc acc act etc aca gtc tec teg 423
20 Asp Cys Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser
130 135 140
<210> 155
25 <211> 141
<212> PRT
<213> Mus musculus
<400> 155
30 Met Val Leu Ala Ser Ser Thr Thr Ser Me His Thr Met Leu Leu Leu
15 10 15
Leu Leu Met Leu Ala Gin Pro Ala Met Ala Glu Val Lys Leu Val Glu
20 25 30
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Arg Lys Leu Ser Cys
137
JP 2003-415746
35 40 45
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser Trp Val Arg
50 55 60
Gin Thr Pro Ala Lys Arg Leu Glu Trp Val Ala Thr He Ser Ser Gly
65 70 75 80
Ser Ser Thr lie Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Me
10 85 90 95
Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe Leu Gin Met Thr Ser Leu
100 105 110
15 Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Trp Phe Leu
115 120 125
Asp Cys Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser
130 135 140
20
<210> 156
<211> 357
<212> DNA
25 <213> Mus musculus
<220>
<221> CDS
<222> (1) . . (357)
30 <223>
<400> 1 56
gat att gtg etc acc caa tct cca get tct ttg get gtg tct eta ggg 48
Asp He Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
35 1 5 10 15
138 JP 2003-415746
20
cag agt gtc acc ate tec tgc aga gee agt gaa agt gtt gaa tat tat 96
Gin Ser Val Thr lie Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
5 ggc act agt tta atg cag tgg tac caa cag aaa cca gga cag cca ccc 144
Gly Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro
35 40 45
aaa etc etc ate tat ggt gca tec aac gta gaa tct ggg gtc cct gee 192
10 Lys Leu Leu Me Tyr Gly Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
30 <210> 157
<211> 119
<212> PRT
<213> Mus musculus
240
agg ttt agt ggc agt ggg tct ggg aca gac ttc age etc aac ate cat
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Me His
15 65 70 75 80
cct gtg gag gag gat gat att gca atg tat ttc tgt cag caa agt agg 288
Pro Val Glu Glu Asp Asp Me Ala Met Tyr Phe Cys Gin Gin Ser Arg
85 90 95
aag gtt ccg tgg acg ttc ggt gga ggc acc aag ctg gaa ata aag gac 336
Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Me Lys Asp
100 105 110
25 tac aag gat gac gac gat aag 357
Tyr Lys Asp Asp Asp Asp Lys
115
35 <400> 157
Asp Me Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
139
JP 2003-415746
10
15
Gin Ser Val Thr He Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro
35 40 45
Lys Leu Leu lie Tyr Gly Ala Ser Asn Val Glu Ser Gly Val Pro Ala
10 50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Me His
65 70 75 80
15 Pro Val Glu Glu Asp Asp lie Ala Met Tyr Phe Cys Gin Gin Ser Arg
85 90 95
Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Me Lys Asp
100 105 110
20
Tyr Lys Asp Asp Asp Asp Lys
115
25 <210> 158
<211> 432
<212> DNA
<213> Mus musculus
30 <220>
<221> CDS
<222> (1) . . (432)
<223>
35 <400> 158
atg gtt ctt gcc age tct acc acc age ate cac acc atg ctg etc ctg
48
140 JP 2003-415746
Met Val Leu Ala Ser Ser Thr Thr Ser Me His Thr Met Leu Leu Leu
1
10
15
etc ctg atg ctg gec cag ccg gec atg gcg cag gtt cag etc cag caa
5 Leu Leu Met Leu Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Gin
20
25
30
96
tct gga cct gag ctg gtg aag cct ggg gec tea gtg aag att tec tgc
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys lie Ser Cys
10 35 40 45
144
15
aag get tct ggc tat gca ttc agt age tec tgg atg aac tgg atg aag
Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser Trp Met Asn Trp Met Lys
50 55 60
cag agg cct gga aag ggt ctt gag tgg att ggg egg att tat cct gga
Gin Arg Pro Gly Lys Gly Leu Glu Trp Me Gly Arg Me Tyr Pro Gly
65 70 75 80
192
240
20 gat gga gat act aac tac aat ggg aag ttc aag ggc aag gec aca ctg
Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu
85 90 95
288
act gca gac aaa tec tec age aca gec tac atg caa etc age age ctg
25 Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu
100 105 110
336
aca tct gag gac tct gcg gtc tac ttc tgt gca aga gcg agg aaa act
Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Ala Arg Lys Thr
30 115 120 125
384
tec tgg ttt get tac tgg ggc caa ggg act ctg gtc act gtc tct gcg
Ser Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala
130 135 140
432
35
141 JP 2003-415746
<210> 159
<211> 144
<212> PRT
<213> Mus musculus
<400> 1 59
Met Val Leu Ala Ser Ser Thr Thr Ser Me His Thr Met Leu Leu Leu
15 10 15
10 Leu Leu Met Leu Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Gin
20 25 30
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys lie Ser Cys
35 40 45
15
Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser Trp Met Asn Trp Met Lys
50 55 60
Gin Arg Pro Gly Lys Gly Leu Glu Trp Me Gly Arg Me Tyr Pro Gly
20 65 70 75 80
Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu
85 90 95
25 Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu
100 105 110
Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Ala Arg Lys Thr
115 120 125
30
Ser Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala
130 135 140
35 <210> 160
<211> 345
142 JP 2003-415746
25
<212> DNA
<213> Mus musculus
<220>
5 <221> CDS
<222> (1 ) . . (345)
<223>
<400> 1 60
10 gac att gtg ttg aca cag tct caa aaa ttc atg tec aca tea gta gga 48
Asp Me Val Leu Thr Gin Ser Gin Lys Phe Met Ser Thr Ser Val Gly
15 10 15
gac agg gtc age ate age tgc aag gee agt cag aat gtg ggt aat att 96
15 Asp Arg Val Ser Me Ser Cys Lys Ala Ser Gin Asn Val Gly Asn Me
20 25 30
ata gec tgg tat caa cag aaa cca ggg caa tct cct aaa gca ctg att 144
Me Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Ala Leu Me
20 35 40 45
tac ttg gca tec tac egg tac agt gga gtc cct gat cgc ttc aca ggc 192
Tyr Leu Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
agt gga tct ggg aca gat ttc act etc ace att agt aat gtg cag tct 240
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Me Ser Asn Val Gin Ser
65 70 75 80
30 gaa gac ttg gca gag tat ttc tgt cag caa tat age age tct ccg etc 288
Glu Asp Leu Ala Glu Tyr Phe Cys Gin Gin Tyr Ser Ser Ser Pro Leu
85 90 95
acg ttc ggt get ggg acc aag ctg gaa ata aag gac tac aag gat gac 336
35 Thr Phe Gly Ala Gly Thr Lys Leu Glu Me Lys Asp Tyr Lys Asp Asp
100 105 110
143
JP 2003-415746
15
gac gat aag 345
Asp Asp Lys
115
<210> 161
<211> 115
<212> PRT
10 <213> Mus musculus
<400> 161
Asp lie Val Leu Thr Gin Ser Gin Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser lie Ser Cys Lys Ala Ser Gin Asn Val Gly Asn Me
20 25 30
e Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Ala Leu lie
20 35 40 45
Tyr Leu Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
25 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Asn Val Gin Ser
65 70 75 80
30
Glu Asp Leu Ala Glu Tyr Phe Cys Gin Gin Tyr Ser Ser Ser Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu lie Lys Asp Tyr Lys Asp Asp
100 105 110
Asp Asp Lys
35 115
144 JP 2003-415746
<210> 162
<211> 116
<212> PRT
5 <213> Mus musculus
<400> 162
Asp Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin
15 10 15
10
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser lie Thr Ser Asp
20 25 30
Tyr Ala Trp Ser Trp I le Arg Gin Leu Pro Gly Asn Lys Leu Glu Trp
15 35 40 45
Met Gly Tyr Me Thr Tyr Ser Gly Tyr Ser Me Tyr Asn Pro Ser Leu
50 55 60
20 Lys Ser Arg I le Ser I le Ser Arg Asp Thr Ser Lys Asn Gin Leu Phe
65 70 75 80
Leu Gin Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
25
Val Gly Gly Tyr Asp Asn Met Asp Tyr Trp Gly Gin Gly Thr Ser Val
100 105 110
Thr Val Ser Ser
30 115
<210> 163
<211> 108
35 <212> PRT
<213> Mus musculus
145 JP 2003-415746
<400> 163
Gin He Val Leu Thr Gin Ser Pro Ala Me Met Ser Ala Ser Pro Gly
15 10 15
Glu Lys Val Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser
20 25 30
His Leu Tyr Trp Tyr Gin Gin Lys Pro Gly Ser Ser Pro Lys Leu Trp
10 35 40 45
e Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
50 55 60
15 Gly Ser Gly Ser Gly Thr Ser. Tyr Ser Leu Thr lie Ser Asn Met Glu
65 70 75 80
Thr Glu Asp Ala Ala Ser Tyr Phe Cys His Gin Trp Ser Ser Tyr Pro
85 90 95
20
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Me Lys
100 105
25 <210> 164
<211> 1924
<212> DNA
<213> Macaca fascicular is
30 <220>
<221> CDS
<222> (11).. (1918)
<223>
35 <400> 164
gaattccacc atg ccc tec tgg gec etc ttc atg gtc acc tec tgc etc
49
146
JP 2003-415746
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu
1 5 10
etc ctg gec cct caa aac ctg gec caa gtc age age caa gat gtc tec
5 Leu Leu Ala Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser
15 20 25
97
ttg ctg gec teg gac tea gag ccc ctg aag tgt ttc tec cga aca ttt
Leu Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe
10 30 35 40 45
145
15
gag gac etc act tgc ttc tgg gat gag gaa gag gca gca ccc agt ggg
Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly
50 55 60
aca tac cag ctg ctg tat gec tac ccg ggg gag aag ccc cgt gec tgc
Thr Tyr Gin Leu Leu Tyr Ala Tyr Pro Gly Glu Lys Pro Arg Ala Cys
65 70 75
193
241
20 ccc ctg agt tct cag age gtg ccc cgc ttt gga acc cga tac gtg tgc
Pro Leu Ser Ser Gin Ser Val Pro Arg Phe Gly Thr Arg Tyr Val Cys
80 85 90
289
cag ttt cca gec cag gaa gaa gtg cgt etc ttc tct ccg ctg cac etc
25 Gin Phe Pro Ala Gin Glu Glu Val Arg Leu Phe Ser Pro Leu His Leu
95 100 105
337
tgg gtg aag aat gtg ttc eta aac cag act cag att cag cga gtc etc
Trp Val Lys Asn Val Phe Leu Asn Gin Thr Gin He Gin Arg Val Leu
30 110 115 120 125
385
35
ttt gtg gac agt gta ggc ctg ccg get ccc ccc agt ate ate aag gee
Phe Val Asp Ser Val Gly Leu Pro Ala Pro Pro Ser Me Me Lys Ala
130 135 140
atg ggt ggg age cag cca ggg gaa ctt cag ate age tgg gag gee cca
433
481
147
JP 2003-415746
Met Gly Gly Ser Gin Pro Gly Glu Leu Gin He Ser Trp Glu Ala Pro
145 150 155
get cca gaa ate agt gat ttc ctg agg tac gaa etc cgc tat ggc ccc
5 Ala Pro Glu Me Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro
160 165 170
529
aaa gat etc aag aac tec act ggt ccc acg gtc ata cag ttg ate gec
Lys Asp Leu Lys Asn Ser Thr Gly Pro Thr Val Me Gin Leu Me Ala
10 175 180 185
577
15
aca gaa acc tgc tgc cct get ctg cag agg cca cac tea gee tct get
Thr Glu Thr Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala
190 195 200 205
ctg gac cag tct cca tgt get cag ccc aca atg ccc tgg caa gat gga
Leu Asp Gin Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly
210 215 220
625
673
20 cca aag cag acc tec cca act aga gaa get tea get ctg aca gca gtg
Pro Lys Gin Thr Ser Pro Thr Arg Glu Ala Ser Ala Leu Thr Ala Val
225 230 235
721
ggt gga age tgc etc ate tea gga etc cag cct ggc aac tec tac tgg
25 Gly Gly Ser Cys Leu Me Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp
240 245 250
769
ctg cag ctg cgc age gaa cct gat ggg ate tec etc ggt ggc tec tgg
Leu Gin Leu Arg Ser Glu Pro Asp Gly Me Ser Leu Gly Gly Ser Trp
30 255 260 265
817
35
gga tec tgg tec etc cct gtg act gtg gac ctg cct gga gat gca gtg
Gly Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val
270 275 280 285
gca att gga ctg caa tgc ttt acc ttg gac ctg aag aat gtt acc tgt
865
913
148 JP 2003-415746
Ala lie Gly Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys
290 295 300
caa tgg cag caa gag gac cat get agt tec caa ggt ttc ttc tac cac 961
5 Gin Trp Gin Gin Glu Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His
305 310 315
age agg gca egg tgc tgc ccc aga gac agg tac ccc ate tgg gag gac 1009
Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro I le Trp Glu Asp
10 320 325 330
tgt gaa gag gaa gag aaa aca aat cca gga tta cag acc cca cag ttc 1057
Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe
335 340 345
15
tct cgc tgc cac ttc aag tea cga aat gac age gtt att cac ate ctt 1105
Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser Val Me His Me Leu
350 355 360 365
20 gtg gag gtg acc aca gec ctg ggt get gtt cac agt tac ctg ggc tec 1153
Val Glu Val Thr Thr Ala Leu Gly Ala Val His Ser Tyr Leu Gly Ser
370 375 380
cct ttc tgg ate cac cag get gtg cgc etc ccc acc cca aac ttg cac 1201
25 Pro Phe Trp Me His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His
385 390 395
tgg agg gag ate tec age ggg cat ctg gaa ttg gag tgg cag cac cca 1249
Trp Arg Glu Me Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro
30 400 405 410
tea tec tgg gca gee caa gag acc tgc tat caa etc cga tac aca gga 1297
Ser Ser Trp Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly
415 420 425
35
gaa ggc cat cag gac tgg aag gtg ctg gag ccg cct etc ggg gee cga 1345
149
JP 2003-415746
Glu Gly His Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg
430 435 440 445
gga ggg acc ctg gag ctg cgc ccg cga tct cgc tac cgt tta cag ctg 1393
5 Gly Gly Thr Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu
450 455 460
cgc gcc agg etc aat ggc ccc acc tac caa ggt ccc tgg age teg tgg 1441
Arg Ala Arg Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp
10 465 470 475
teg gac cca get agg gtg gag acc gcc acc gag acc gcc tgg att tec 1489
Ser Asp Pro Ala Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser
480 485 490
15
ttg gtg acc get ctg ctg eta gtg ctg ggc etc age gcc gtc ctg ggc 1537
Leu Val Thr Ala Leu Leu Leu Val Leu Gly Leu Ser Ala Val Leu Gly
495 500 505
20 ctg ctg ctg ctg agg tgg cag ttt cct gca cac tac agg aga ctg agg 1585
Leu Leu Leu Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg
510 515 520 525
cat gcc ctg tgg ccc tea ctt cca gat ctg cac cga gtc eta ggc cag 1633
25 His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin
530 535 540
tac ctt agg gac act gca gcc ctg agt ccg ccc aag gcc aca gtc tea 1681
Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser
30 545 550 555
gat acc tgt gaa gaa gtg gaa ccc age etc ctt gaa ate etc ccc aag 1729
Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu lie Leu Pro Lys
560 565 570
35
tec tea gag agg act cct ttg ccc ctg tgt tec tec cag tec cag atg 1777
150
JP 2003-415746
Ser Ser 61 u Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ser Gin Met
575 580 585
gac tac cga aga ttg cag cct tct tgc ctg ggg acc atg ccc ctg tct 1825
5 Asp Tyr Arg Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser
590 595 600 605
gtg tgc cca ccc atg get gag tea ggg tec tgc tgt acc acc cac att 1873
Val Cys Pro Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His lie
10 610 615 620
gee aac cat tec tac eta cca eta age tat tgg cag cag cct tga 1918
Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
15
gtcgac 1 924
<210> 165
20 <211> 635
<212> PRT
<213> Macaca fascicular is
<400> 1 65
25 Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala
1 5 10 15
Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala
20 25 30
30
Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu
35 40 45
Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin
35 50 55 60
151 JP 2003-41 5746
Leu Leu Tyr Ala Tyr Pro Gly Glu Lys Pro Arg Ala Cys Pro Leu Ser
65 70 75 80
Ser Gin Ser Val Pro Arg Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro
5 85 90 95
Ala Gin Glu Glu Val Arg Leu Phe Ser Pro Leu His Leu Trp Val Lys
100 105 110
10 Asn Val Phe Leu Asn Gin Thr Gin He Gin Arg Val Leu Phe Val Asp
115 120 125
Ser Val Gly Leu Pro Ala Pro Pro Ser Me He Lys Ala Met Gly Gly
130 135 140
15
Ser Gin Pro Gly Glu Leu Gin He Ser Trp Glu Ala Pro Ala Pro Glu
145 150 155 160
lie Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro Lys Asp Leu
20 165 170 175
Lys Asn Ser Thr Gly Pro Thr Val lie Gin Leu lie Ala Thr Glu Thr
180 185 190
25 Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala Leu Asp Gin
1 95 200 205
Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly Pro Lys Gin
210 215 220
30
Thr Ser Pro Thr Arg Glu Ala Ser Ala Leu Thr Ala Val Gly Gly Ser
225 230 235 240
Cys Leu He Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp Leu Gin Leu
35 245 250 255
152
JP 2003-415746
Arg Ser Glu
Ser Leu Pro
5 275
Leu Gin Cys
290
10 Gin Glu Asp
305
Arg Cys Cys
15
Glu Glu Lys
His Phe Lys
20 355
Thr Thr Ala
370
25 lie His Gin
385
He Ser Ser
30
Ala Ala Gin
Gin Asp Trp
35 435
Pro Asp Gly Me
260
Val Thr Val Asp
Phe Thr Leu Asp
295
His Ala Ser Ser
310
Pro Arg Asp Arg
325
Thr Asn Pro Gly
340
Ser Arg Asn Asp
Leu Gly Ala Val
375
Ala Val Arg Leu
390
Gly His Leu Glu
405
Glu Thr Cys Tyr
420
Lys Val Leu Glu
Ser Leu Gly Gly Ser
265
Leu Pro Gly Asp Ala
280
Leu Lys Asn Val Thr
300
Gin Gly Phe Phe Tyr
315
Tyr Pro I le Trp Glu
330
Leu Gin Thr Pro Gin
345
Ser Val I le His I le
360
His Ser Tyr Leu Gly
380
Pro Thr Pro Asn Leu
395
Leu Glu Trp Gin His
410
Gin Leu Arg Tyr Thr
425
Pro Pro Leu Gly Ala
440
Trp Gly Ser Trp
270
Val Ala lie Gly
285
Cys Gin Trp Gin
His Ser Arg Ala
320
Asp Cys Glu Glu
335
Phe Ser Arg Cys
350
Leu Val Glu Val
365
Ser Pro Phe Trp
His Trp Arg Glu
400
Pro Ser Ser Trp
415
Gly Glu Gly His
430
Arg Gly Gly Thr
445
153
JP 2003-415746
Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu Arg Ala Arg
450 455 460
Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp Ser Asp Pro
5 465 470 475 480
Ala Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser Leu Val Thr
485 490 495
10 Ala Leu Leu Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu
500 505 510
Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu
515 520 525
15
Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin Tyr Leu Arg
530 535 540
Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp Thr Cys
20 545 550 555 560
Glu Glu Val Glu Pro Ser Leu Leu Glu He Leu Pro Lys Ser Ser Glu
565 570 575
25 Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ser Gin Met Asp Tyr Arg
580 585 590
Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser Val Cys Pro
595 600 605
30
Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Me Ala Asn His
610 615 620
Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
35 625 630 635
154 JP 2003-415746
<210> 166
<211> 24
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized sequence
10 <400> 166
caggggccag tggatagact gatg 24
<210> 167
15 <211> 23
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized sequence
<400> 167
gctcactgga tggtgggaag atg 23
25
30
<210> 168
<211> 30
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 1 68
35 tagaattcca ccatggaatg gcctttgatc
30
JP 2003-415746
<210> 169
<211> 56
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 169
agcctgagtc atcacaatat ccgatccgcc tccacctgca gagacagtga ccagag 56
<210> 170
15 <211> 56
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 170
actctggtca ctgtctctgc aggtggaggc ggatcggata ttgtgatgac tcaggc 56
<210> 171
<211> 60
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 171
35 attgcggccg cttatcactt atcgtcgtca tccttgtagt cttttatttc cagcttggtc 60
156 JP 2003-415746
<210> 172
<211> 8
<212> PRT
5 <213> Artificial
<220>
<223> an artificially synthesized FLAG tag sequence
10 <400> 172
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
15 <210> 173
<211> 85
<212> DNA
<213> Artificial
20 <220>
<223> an artificially synthesized primer sequence
<400> 1 73
tagaattcca ccatggaatg gcctttgatc tttctcttcc tcctgtcagg aactgcaggt 60
25
gtccactccc aggttcagct gcagc 85
<210> 174
30 <211> 82
<212> DNA
<213> Artificial
<220>
35 <223> an artificially synthesized primer sequence
157 JP 2003-415746
5
20
25
<400> 1 74
tggtcactgt ctctgcaggt ggtggtggtt cgggtggtgg tggttcgggt ggtggcggat 60
cggatattgt gatgactcag gc 82
<210> 175
<211> 82
<212> DNA
10 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
1 5 <400> 1 75
tgagtcatca caatatccga tccgccacca cccgaaccac caccacccga accaccacca 60
cctgcagaga cagtgaccag ag 82
<210> 176
<211> 25
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 1 76
30 caggttcagc tgcagcagtc tggac 25
<210> 177
<211> 81
35 <212> DNA
<213> Artificial
158 JP 2003-415746
<220>
<223> an artificially synthesized primer sequence
5 <400> 177
gctgcagctg aacctgcgat ccaccgcctc ccgaaccacc accacccgat ccaccacctc 60
cttttatttc cagcttggtc c 81
10
<210> 178
<211> 38
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 1 78
20 gcccagccgg ccatggcgga kgtrmagctt caggagtc 38
<210> 179
<211> 38
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 1 79
gcccagccgg ccatggcgga ggtbcagctb cagcagtc 38
35 <210> 180
<211> 38
159 JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 1 80
gcccagccgg ccatggcgca ggtgcagctg aagsastc 38
10
<210> 181
<21 1 > 38
<212> DNA
<213> Artificial
15
<220>
<223> an artificial ly synthesized primer sequence
<400> 1 81
20 gcccagccgg ccatggcgga ggtccarctg caacartc 38
<210> 182
<211> 38
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 1 82
gcccagccgg ccatggcgca ggtycagctb cagcartc 38
35
<210> 183
<211> 38
160
JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 1 83
gcccagccgg ccatggcgca ggtycarctg cagcagtc 38
10
<210> 184
<211> 38
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 184
20 gcccagccgg ccatggcgca ggtccacgtg aagcagtc 38
<210> 185
<211> 38
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 185
gcccagccgg ccatggcgga ggtgaasstg gtggaatc 38
35 <210> 186
<211> 38
161 JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 186
gcccagccgg ccatggcgga vgtgawgytg gtggagtc 38
10
<210> 187
<211> 38
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 187
20 gcccagccgg ccatggcgga ggtgcagskg gtggagtc 38
<210> 188
<211> 38
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 188
gcccagccgg ccatggcgga kgtgcamctg gtggagtc 38
35 <210> 189
<211> 38
162
JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 189
gcccagccgg ccatggcgga ggtgaagctg atggartc 38
10
<210> 190
<211> 38
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 1 90
20 gcccagccgg ccatggcgga ggtgcarctt gttgagtc 38
<210> 191
<211> 38
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 191
gcccagccgg ccatggcgga rgtraagctt ctcgagtc 38
35 <210> 192
<211> 38
163 JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 192
gcccagccgg ccatggcgga agtgaarstt gaggagtc 38
<210> 193
<211> 40
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 193
20 gcccagccgg ccatggcgca ggttactctr aaagwgtstg 40
<210> 194
<211> 38
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 1 94
gcccagccgg ccatggcgca ggtccaactv cagcarcc 38
35 <210> 195
<211> 38
164
JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 195
gcccagccgg ccatggcgga tgtgaacttg gaagtgtc 38
10
<210> 196
<211> 38
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 196
20 gcccagccgg ccatggcgga ggtgaaggtc atcgagtc 38
<210> 197
<211> 36
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 1 97
ggagccgccg ccgcccgagg aaacggtgac cgtggt 36
35 <210> 198
<211> 36
165 JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence .
<400> 1 98
ggagccgccg ccgcccgagg agactgtgag agtggt 36
10
<210> 199
<211> 36
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 199
20 ggagccgccg ccgcccgcag agacagtgac cagagt 36
<210> 200
<211> 36
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 200
ggagccgccg ccgcccgagg agacggtgac tgaggt 36
35 <210> 201
<211> 35
166 JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 201
ggcggcggcg gctccgayat ccagctgact cagcc 35
10
<210> 202
<211> 35
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 202
20 ggcggcggcg gctccgayat tgttctcwcc cagtc 35
<210> 203
<211> 35
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 203
ggcggcggcg gctccgayat tgtgmtmact cagtc 35
35 <210> 204
<211> 35
167 JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 204
ggcggcggcg gctccgayat tgtgytraca cagtc 35
10
<210> 205
<211> 35
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 205
20 ggcggcggcg gctccgayat tgtratgacm cagtc 35
<210> 206
<211> 35
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 206
ggcggcggcg gctccgayat tmagatramc cagtc 35
35 <210> 207
<211> 35
168
JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 207
ggcggcggcg gctccgayat tcagatgayd cagtc 35
10
<210> 208
<211> 35
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 208
20 ggcggcggcg gctccgayat ycagatgaca cagac 35
<210> 209
<211> 35
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 209
ggcggcggcg gctccgayat tgttctcawc cagtc 35
35 <210> 210
<211> 35
169
JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 210
ggcggcggcg gctccgayat tgwgctsacc caatc 35
10
<210> 211
<211> 35
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 211
20 ggcggcggcg gctccgayat tstratgacc carte 35
<210> 212
<211> 35
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 212
ggcggcggcg gctccgayrt tktgatgacc carac 35
35 <210> 213
<211> 35
170
JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 213
ggcggcggcg gctccgayat tgtgatgacb cagkc 35
10
<210> 214
<211> 35
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 214
20 ggcggcggcg gctccgayat tgtgataacy cagga 35
<210> 215
<211> 35
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 215
ggcggcggcg gctccgayat tgtgatgacc cagwt 35
35 <210> 216
<211> 35
171 JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 216
ggcggcggcg gctccgayat tgtgatgaca caacc 35
10
<210> 217
<211> 35
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 217
20 ggcggcggcg gctccgayat tttgctgact cagtc 35
<210> 218
<211> 38
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
30
<400> 218
ggcggcggcg gctccgatgc tgttgtgact caggaatc 38
35 <210> 219
<211> 36
172
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 219
ggaattcggc ccccgaggcc ttgatttcca gcttgg
<210> 220
<211> 36
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 220
ggaattcggc ccccgaggcc tttatttcca gcttgg
<210> 221
<211> 36
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 221
ggaattcggc ccccgaggcc tttatttcca actttg
<210> 222
<211> 36
173
JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized primer sequence
<400> 222
ggaattcggc ccccgaggcc ttcagctcca gcttgg 36
10
<210> 223
<211> 39
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized primer sequence
<400> 223
20 ggaattcggc ccccgaggcc cctaggacag tcagtttgg 39
<210> 224
<211> 27
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized sequence
30
<400> 224
ttactcgcgg cccagccggc catggcg 27
35 <210> 225
<211> 17
174 JP 2003-415746
<212> DNA
<213> Artificial
<220>
5 <223> an artificially synthesized sequence
<400> 225
ggaattcggc ccccgag 17
10
<210> 226
<211> 20
<212> DNA
<213> Artificial
15
<220>
<223> an artificially synthesized sequence
<400> 226
20 tcacttacag gctctctact 20
<210> 227
<211> 20
25 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized sequence
30
<400> 227
caggtggggt ctttcattcc 20
175
JP 2003-415746
[Document Name] Drawings
[Fig- 1]
EcoRI
dbVB22B
IVHi
VL
SIGNAL SEQUENCE LINKER (Gly 4 Ser)
-4 B
pvu n
4
FLAG SEQUENCE
" r°
Notl
PCR RECOMBINATION
scVB22B
VH
VL
SIGNAL SEQUENCE
LINKER (Gly 4 Ser) 3
Pvu n
1
PCR AMPLIFICATION
FLAG SEQUENCE
F
Pvu n
VH
VL
lis
H
i
CLONING INTO
Pvull SITE
Pvu D
VB22B
sc(Fv)2 SIGNAL SEQUENCE LINKER(Gly 4 Ser) 3
VL
L INKER (Gly 4 Ser) 3
LINKER(Gly 4 Ser) 3
FLAG SEQUENCE
176
JP 2003-415746
[Fig. 2]
CHO-HUMAN MpI CHO-MONKEY Mpl
FITC LOG FITC LOG
CHO-MOUSE MpI CHO
FITC LOG FITC LOG
177
JP 2003-415746
[Fig- 3]
1,6
0.000 0.001 0.010 0.100 1.000 10.000
CONCENTRATION [nM]
[Fig. 4]
1.8
0.000 0.Q01 0.010 0.100 1.000 10.000
CONCENTRATION [nM]
178
JP 2003-415746
0.001
0.01 0.1 1
CONCENTRATION (nM)
10
100
[Fig. 6]
VA7
VA130
VA259
VB17B
VB12B
VB140
VB33
VB45B
VB8B
VB115
VB14B
VB22B
VB16
VB157
VB4B
VB51
VA7
VA130
VA259
VB17B
VB12B
VB140
VB33
VB45B
VB8B
VB115
VB14B
VB22B
VB16
VB157
VB4B
VB51
CDR1
CDR2
Q VQLQQS G PE L VK PG AS VK ISC KAS G Y A FS
QVQLQQSGPELVKPGASVKISCKASGYAFS
QVQLQQS G PE L VK PGAS VKI S CKAS G Y A FS
QVQLQQSGPELVKPGASVKISCKASGYTFS
QVQLQQSGPELVKPGASVKISCKASGYAFS
QVQLQQS GPELVKPGAS VKI SCRAFGYAFS
QVQLQQPGAELVKPGASVKLSCKASGYTFT
QVQLQQSGPELVKPGASVKISCKASGYAFS
QVQLQQSGPELVKPGASVKISCKASGYAFS
QVQLQQSGPELVKPGASVKISCKASGYAFS
QVQLQQS G PE L LN PG AS VK I S C KAS G Y A FS
QVQLQQS G PE L VK PGAS VK I S CKAS G YA FT
QVQLQQPGTELVRPGASVKLSCKASGYTFT
QVQLQQPGAELVKPGASVKLSCKASGYTFT
QVQLQQS G PEL VK PGAS VKI S CKAS GY AFT
QVQLQQSGPELVKPGASVKISCKASGYAFS
SSWMN
SSWMN
SSWMN
SSWMN
RSWMN
NSWMN
NYWVN
SSWMN
TSWMN
SSWMN
RSWMN
NSWMN
DYWVN
DYWMN
NSWMN
NSWMN
WVKQRPGKGLEW I G
WVKQRPGKGLEWIG
WVKQRPGKGLEW IG
WVKQRPGKGLEWIG
WVKQRPGKGLEWIG
WVKQRPGKGLEWIG
WVKQRPGRGLEWIG
WVKQRPGKGLEWIG
WVKQRPGKGLEWIG
WVKQRPGKGPEWIG
WVKQRPGKGLEWIG
WVKQRPGKGLEWIG
WVKQRPGRGLEWIG
WVKQRPGRGLEW IG
WVRQRPGKGLEWIG
WVNQRPGKGLEWIG
RTY PGDGDTN YNGKFKG
RIYPGDGDTNYNGKFKG
RIYPGDGETN YNGKFKG
RIYPGDGDTNYNGKFKG
RIYPGDGDTNYNGKFKG
RI Y PGDGETNNNGKFKG
RIHPSDSETHCNQKFKR
RIY PGDGETNNNGKFKG
RIYPGDGEAN YNGKFKG
RIYPGDGETN YNGKFKG
RIYPGDGETNYNGKFKG
RIYPGDGETI YNGKFRV
RIHPYDSETHYNQKFKN
RIHPFDSETHCSQKFKN
RIYPGDGETI YNGKFRV
RIYPGDGDTIYNGNFKG
CDR3
KAT LTADKS S S TAYMQLS SLTSE DSAVY FCAR
KAT LTADKS S S T AY I QLS S LT S E DS AVY FCAR
KAT LTADKS S NT A YMQLS SLTSE DSAVY FCAR
KAT LTADKS SSTAYMQLSSLTSEDS AVY FCAS
KAT LTADKS SSTAYMQLSSLTSEDS AVY FCAS
KATLTADKSSSTAYMQLS SLTSE DSAVY FCAR
KATLTVNKSS STAY IQLHSLTS EDS AVY YCTS
KAT LTADKS S T TAYMQLS SLTSE DSAVY FCAR
KAT LTADKS S S S A YMQLS S LTSE DSAVY FCAR
KATLT ADKS S STVYMQLS S LTSE DSAVY FCAR
KAT LTADKS STTAYMQFS SLTSE DSAVY FCAR
KAT LTADKS SSTAYMDIS SLTSE DSAVY FCAR
KATLTVDKSSSTAYIQLSSLTSEDSAVYYCAS
KATLTVDKS SNT AY I QFS S LTSE DS AVY YCS S
KAT LTADKS S S TAYME I S S LT S E DSAVY FCAR
KATLTADKSSSIAYMQLSSLTSEDSAVYFCTS
GWILADGG YSFAY
WGQGTLVTVSA
GYAD —
— YSFAY
WGQGTLVTVSA
GFGD —
— YSFAY
WGQGTLVTVSA
GYAD—
— YSFAY
WGQGTLVTVSA
GYDD —
— YSFAY
WGQGTLVTVSA
GYGD —
— YSFAY
WGQGTLVTVSA
GGW
FAY
WGQGTLVTVSA
GYGD —
— YSFAY
WGQGTLVTVSA
GYGD —
— YSFAY
WGQGTLVTVSA
GYGD —
— YSFAY
WGQGTLVTVSA
GDGD —
— YSFAY
WGQGTLVTVSA
GYDD —
— YSFAY
WGQGTLVTVSA
GGW
FAS
WGQGTLVTVSA
GGW
FAY
WGQGTLVTVSA
GYDD —
— YSFAY
WGQGTLVTVSA
GYDD —
— YSFAY
WGQGTLVTVSA
179 JP 2003-415746
[Fig. 7]
VA7
VA130
VA259
VB17B
VB12B
VB140
VB33
VB4 5B
VB8B
VB115
VB14B
VB22B
VB16
VB157
VB4B
VB51
VA7
VA130
VA2 5 9
VB17B
VB12B
VB140
VB33
VB4 5B
VB8B
VB115
VB14B
VB22B
VB16
VB157
VB4B
VB51
DIVMTQAAPSIPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
D I VMTQAAPS VPVT PGES VS ISC
D I VMTQAAPS VPVT PGESVS ISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
D I VMTQAAP S VPVT PGE S VS ISC
DIVMTQAAPSVPVTPGESVSISC
D I VMTQAAP S I PVT PGE S VS ISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVSVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSLPVTPGESVSISC
CDR1
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLYSNGNI YLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLYSNGNTYLY
RSSKSLLYSNGNI YLY
RSSKSLLHNNGNTYLY
RSSKSLLHSNGNTYLY
WFLQRPGQS PQLLI Y
WFLQRPGQS PQLL I Y
WFLQRPGQS PQLLI Y
WFLQRPGQS PQLL I Y
WFLQRPGQS PQLL I Y
WFLQRPGQS PQLL I Y
WFLQRPGQS PQLL I Y
WFLQRPGQS PQLL I Y
WFMQRPGQS PQLLI Y
WFLQRPGQS PQLL I Y
WFLQRPGQS PQLLI Y
WFLQRPGQS PQLL I Y
WFLQRPGQ S PQLL I Y
WFLQRPGQ S PQLL I Y
WFLQRPGQ S PQLL I Y
WFLQRPGQS PQLL I Y
CDR3
GVPDRFSGSGSGTAFTLRISRVEAEDVGI YYC
GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC
GAPDRFSGSGSGTAFTLRISRVETEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGAAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGAAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLT I S SVEAEDVGVYYC
GVPDRFSGSGSGTAFTLKI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
MQHLEYPFT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHVEYPYT
MQHLEYPYT
MQHLEYPYT
MQHIEYPFT
MQHLEYPYT
MQHLEYPYT
MQHIEYPFT
MQHLEYPYT
FGTGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
CDR2
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
180
JP 2003-415746
[Fig- 8]
CHO-HUMAN MpI CHO-MONKEY Mpl
FITC LOG FITC LOG
CHO-MOUSE MpI CHO
FITC LOG FITC LOG
181 JP 2003-415746
[Fig. 9]
2
_ 18
i 1.6
LO
50 1.4
E
c 1.2
10
1
LU
o 0.8
2
m 0.6 h
0 04
GO
m
< 0.2
0
t*— AB324 DIABODY
AB317 DIABODY
G-TPO
0.000
0.001
0.010 0.100 1.000
CONCENTRATION [nM]
10.000 100.000
[Fig. 10]
0.000
0.001
0.010 0.100 1.000
CONCENTRATION [nM]
10.000 100.000
182
JP 2003-415746
[Fig. 11]
0.000 0.001 0.010 0.100 1.000 10.000 100.000
CONCENTRATION [nM]
183
JP 2003-415746
[Document Name] Abstract
[Abstract]
[Problems to be Solved] An objective of the present invention is to provide novel anti-Mpl
antibodies having TPO-agonistic activity.
[Means for Solving the Problems] Anti-human Mpl antibodies were isolated and purified, and
then anti-human Mpl diabodies and anti-human Mpl sv(Fv) 2 were purified using genetic
engineering techniques. Furthermore, the present inventors succeeded in humanizing
anti-human Mpl sc(Fv)2.
The diabodies and sc(Fv) 2 were assayed for TPO-like agonistic activity, and were found
to have activities higher than those of anti-human Mpl antibodies, or activities equivalent to or
higher than those of naturally-occurring human TPO ligand.
[Selected Drawings] None
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